Caries risk test for predicting and assessing the risk of disease

ABSTRACT

Provided are methods, test devices, and diagnostic kits for predicting, assessing, and diagnosing the risk of a disease using salivary analysis. The method comprises providing a whole (unfractionated) saliva sample from a subject; contacting an aliquot of said saliva with one or more lectins under conditions that allow said one or more lectins to bind to a lectin-binding component of said saliva; detecting the amount of bound lectin; and comparing the amount of bound lectin to the amount known to bind a saliva sample from a control patient, to predict the risk of a disease in the subject. Also provided are methods for reducing the risk of a disease and a method for assessing the risk of the disease at a defined level.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/459,878, filed Apr. 1, 2003, the content of which is incorporatedherein by reference.

FIELD OF THE INVENTION

This present invention provides methods and test devices for predicting,assessing, and diagnosing the risk of a disease using salivary analysis.

BACKGROUND OF THE INVENTION

Insight into a wide range of clinical situations may be gained fromsalivary analysis (See e.g., Mandel, I. D., “Salivary Diagnosis:Promises, Promises,” in Saliva as a Diagnostic Fluid, Malamud et al.,eds., Ann. NY Acad. Sciences 694: 1-10 (1993)). The earliestsialochemical studies on oral fluids examined saliva for specificcomponents that would be diagnostic for various systemic conditions,including gout and rheumatism. (Atkinson, et al., “Guidelines for SalivaNomenclature and Collection,” in Saliva as a Diagnostic Fluid, Ann. NYAcad. Sciences 694: xi-xii (1993)). A recent example utilizes salivaryacetaminophen concentration to assess the gastric emptying rate ofliquids. A number of studies report using saliva specimens formonitoring pharmaceuticals and chemicals, including taxol (Svojanovsky,et al., J. Pharm. Biomed. Anal., 20:549-555 (1999)); caffeine(Akinyinka, et al., Eur. J. Clin. Pharmacol., 56:159-165 (2000)); andthe protease inhibitor, indinavir (Wintergerst, et al., AntimicrobialAgents and Chemotherapy, 44:2572-2574 (2000)).

In general, the analysis of saliva for diagnostic purposes has beendirected towards evaluating systemic disease (e.g., Sjogren's syndrome,cystic fibrosis, HIV infection, etc.), or as a means of determiningsystemic levels of therapeutic drugs such as steroids (Ferguson, J.Dent. Res. 66(2): 420-424 (1987)). There have also been many attempts tomeasure other factors in saliva and then relate them to oral diseases.For example, salivary analysis has also been used to diagnoseperiodontal disease (U.S. Pat. No. 6,063,588 to Lamster; U.S. Pat. No.5,376,532 to Singer, Jr.; U.S. Pat. Nos. 5,866,432 and 5,736,341 toSorsa, et al.; and U.S. Pat. No. 5,756,361 to Winterbottom, et al.).However, the vast majority of studies have not been able to relatefactors in saliva with other common oral diseases such as dental caries.These studies reported values for pH, various ions, macromolecules, andflow rate, but found little evidence of a correlation. The few studiesthat have shown a small amount of correlation were found not tocorrelate with other studies. Thus, there remains a need for asaliva-based test for predicting the risk of oral diseases andassociated diseases that is simple and accurate.

I. Oral Diseases and Associated Diseases

The most common oral diseases are dental caries and the periodontaldiseases. Individuals are vulnerable to dental caries throughout life,affecting 85 percent of adults aged 18 and older. Periodontal diseasesare most often seen in maturity, with the majority of adultsexperiencing some signs and symptoms by the mid-30s. Certain rare formsof periodontal disease affect young people. Oral disorders also includecertain mucosal infections, as well as oral and pharyngeal cancers andprecancerous lesions.

A. Dental Caries

Caries is a unique multifactorial infectious disease (Lenander-Lumikari,et al., Adv. Dent. Res. 14:4047 (December 2000)). Dental caries affectsteeth at all levels and can cause extensive crown mutilations, bacterialdisorders of the periapical tissues, or even loss of the affected dentalelements. Clinically, the disease is characterized by demineralizationof the dental enamel and of the dentin in various stages of progress,until it affects the pulp space. When the lesion passes beyond theenamel-dentin border, a phlogistic reaction of the pulp tissues isconstantly observed, with the formation of reaction dentin in somecases. Approximately 50% of adult individuals have at least fourcaries-related lesions that are treated and require treatment, andapproximately 30% of adult individuals have over 50% of their teethaffected by caries (U.S. Pat. No. 5,830,489 to Valenti, et al.).

The bacterium Streptococcus mutans, or S. mutans, is known to be a primeetiologic agent for the initiation and progression of human dentalcaries, or cavities. S. mutans is one of the primary factors in aciddissolution of the apatite (mineral) component of the enamel then thedentin, or of the cementum then the dentin (Tanzer, J. M., Inter. J.Oral Biol. 22:205-214 (1997)). A strong correlation between theproportion of S. mutans in dental plaque or in saliva relative to otherbacterial species and the presence or risk of future outbreaks of dentalcaries has been documented (Tanzer, J. M., supra). Therefore, S. mutansin plaque or saliva may serve as an index for both caries activity stateand caries risk or susceptibility. These indices play an increasinglyimportant role in the diagnosis and treatment of dental caries (Hume, W.R., J. Dent. Educ. 57:439-443 (1993)).

Present techniques for detecting and quantitatively determining S.mutans include bacterial culture with selective media using either brothor agar plate systems, and polymerase chain reaction techniques (Ellen,R. P., Oral Sci. Rev. 8:3-23 (1976); Igarashi, et al., Oral Microbiol.and Immunol. 11:294-298 (1996); U.S. Pat. No. 5,374,538 to Bratthall;U.S. Pat. No. 4,692,407 to Jordan, et al.). However, each of thesemethods require significant time. (on the order of days), well trainedpersonnel and sophisticated equipment to perform. Consequently, existingtechniques are relatively expensive and time consuming. Moreover, theuse of the titer of S. mutans in the oral cavity as a predictor ofcaries risk is consistently significant only within the first two yearsof age.

Human dental caries may also be detected by changes in translucency,color, hardness or X-ray density of teeth. However, these technologieshave limitations both in specificity and reproducibility. Furthermore,they do not show whether or not the disease is active at a single timepoint (U.S. Pat. No. 6,231,857 to Shi, et al.).

B. Periodontal Diseases

Like dental caries, the periodontal diseases are infections caused bybacteria in the biofilm (dental plaque) that forms on oral surfaces. Thebasic division in the periodontal diseases is between gingivitis, whichaffects the gums, and periodontitis, which may involve all of the softtissue and bone supporting the teeth. Gingivitis and milder forms ofperiodontitis are common in adults. The percentage of individuals withmoderate to severe periodontitis, in which the destruction of supportingtissue may cause the tooth to loosen and fall out, increases with age.

1. Gingivitis

Gingivitis is an inflammation of the gums characterized by a change incolor from normal pink to red, with swelling, bleeding, and oftensensitivity and tenderness. These changes result from an accumulation ofbiofilm along the gingival margins and the immune system's inflammatoryresponse to the release of destructive bacterial products. The earlychanges of gingivitis are reversible with thorough toothbrushing andflossing to reduce plaque. Without adequate oral hygiene, however, theseearly changes can become more severe, with infiltration of inflammatorycells and establishment of a chronic infection. Biofilm on toothsurfaces opposite the openings of the salivary glands often mineralizesto form calculus or tartar, which is covered by unmineralized biofilm—acombination that may exacerbate local inflammatory responses (Mandel, J.Am. Dent. Assoc., 126:573-80 (1995)). A gingival infection can persistfor months or years, yet never progress to periodontitis.

Gingival inflammation does not appear until the biofilm changes from onecomposed largely of gram-positive streptococci (which can live with orwithout oxygen) to one containing gram-negative anaerobes (which cannotlive in the presence of oxygen). Numerous attempts have been made topinpoint which microorganisms in the supragingival (above the gum line)plaque are the culprits in gingivitis. Frequently mentioned organismsinclude Fusobacterium nucleatum, Veillonella parvula, and species ofCampylobacter and Treponema.

Gingival inflammation may be influenced by steroid hormones, occurringas puberty gingivitis, pregnancy gingivitis, and gingivitis associatedwith birth control medication or steroid therapy. The presence ofsteroid hormones in tissues adjacent to biofilm apparently encouragesthe growth of certain bacteria and triggers an exaggerated response tobiofilm accumulation (Caton, “Periodontal diagnosis and diagnosticaids,” in Proceedings of the World Workshop in Clinical Periodontics,American Academy of Periodontology, pp. I-1-22, Princeton, N.J. (1989)).Certain prescription drugs may also lead to gingival overgrowth andinflammation. These include the antiepileptic drug phenytoin(DILANTIN.RTM.), cyclosporin, and various calcium channel blockers usedin heart disease.

2. Adult Periodontitis

The most common form of adult periodontitis is described as general andmoderately progressing. A second form is described as rapidlyprogressing and severe, and is often resistant to treatment. Themoderately progressive adult form is characterized by a gradual loss ofattachment of the periodontal ligament to the gingiva and bone, alongwith loss of the supporting bone. It is most often accompanied bygingivitis (Genco, “Classification of clinical and radiographic featuresof periodontal diseases,” in Contemporary Periodontics, Genco et al.,eds., pp. 63-81, (1990)). It is not necessarily preceded by gingivitis,but the gingivitis-related biofilm often seeds the subgingival plaque.The destruction of periodontal ligament and bone results in theformation of a pocket between the tooth and adjacent tissues, whichharbors subgingival plaque. The calculus formed in the pocket byinflammatory fluids and minerals in adjacent tissues is especiallydamaging (Mandel and Gaffar, J. Chin. Periodontol., 13: 249-57 (1986)).

The severity of periodontal disease is determined through a series ofmeasurements, including the extent of gingival inflammation andbleeding, the probing depth of the pocket to the point of resistance,the clinical attachment loss of the periodontal ligament measured from afixed point on the tooth (usually the cemento-enamel junction), and theloss of adjacent alveolar bone as measured by x-ray (Genco, J.Periodontol., 67(10 Suppl.): 1041-9 (1996)). Severity is determined bythe rate of disease progression over time and the response of thetissues to treatment. Adult periodontitis often begins in adolescencebut is usually not clinically significant until the mid-30s. Prevalenceand severity increase but do not accelerate with age (Beck, Ann.Periodontol., 7(1): 322-57 (1996)).

3. Early-Onset Periodontitis

The forms of periodontitis occurring in adolescents and young adultsgenerally involve defects in neutrophil function (Van Dyke et al;,Infect. Immun. 27(1): 124-31 (1980)). Localized juvenile periodontitis(LJP) mainly affects the first molar and incisor teeth -of teenagers andyoung adults, with rapid destruction of bone but almost no telltalesigns of inflammation and very little supragingival plaque or calculus.Actinobacillus actinomycetemcomitans has been isolated at 90 to 100percent of diseased sites in these patients, but is absent or appears invery low frequency in healthy or minimally diseased sites (Socransky andHaffajee, J. Periodontol., 63(4 Suppl.): 322-31 (1992)). It is possiblethat the bacteria are transmitted among family members through oralcontacts such as kissing or sharing utensils, because the same bacterialstrain appears in affected family members. However, evidence of aneutrophil defect argues for a genetic component. Another organismfrequently associated with LJP is Capnocytophaga ochracea. Neither ofthese bacteria dominate in the generalized adult form of the disease,where Porphyromonas gingivalis is considered of greatest significance(Schenkein and Van Dyke, Periodontol., 6: 7-25 (1994).

Prepubertal periodontitis is rare and may be either general orlocalized. The generalized form begins with the eruption of the primaryteeth and proceeds to involve the permanent teeth. There is severeinflammation, rapid bone loss, tooth mobility, and tooth loss. Thelocalized form of the disease is less aggressive, affecting only someprimary teeth. The infection involves many of the organisms associatedwith periodontitis, but the mix can differ somewhat, with Actinobacillusactinomycetemcomitans, Prevotella intermedia, Eikenella corrodens, andseveral species of Capnocytophaga implicated (Caton, supra). Defects inneutrophil function in both forms of the disease may explain whypatients are highly susceptible to other infections as well (Suzuki,Dent. Clin. North Am., 32(2): 195-216 (1988)).

C. Other Diseases Associated With Oral Disorders

Chronic obstructive pulmonary disease, characterized by obstruction ofairflow due to chronic bronchitis or emphysema and by recurrent episodesof respiratory infection, has been associated with poor oral healthstatus (Hayes et al., Ann. Periodontol. 3(1):257-61 (1998); Scannapiecoet al., Ann. Periondontol., 3(1):251-6 (1998)). A positive relationshipbetween periodontal disease and bacterial pneumonia has also been shown(Scannapieco and Mylotte, J. Periodontol., 67(10 Suppl.): 1114-22(1996)).

Recent studies have also underscored the association of oral infectionswith certain medically important conditions. Increasing data implicateperiodontal disease as a risk factor for cardiovascular diseases such asheart attack and stroke (See e.g., U.S. Pat. No. 6,130,042 to Diehl, etal.; J. Beck, et al., J. Periodontol., 67:1123 (1996)). Epidemiologicstudies indicate that, even after accounting for other known riskfactors for cardiovascular disease, the relative risk attributable toperiodontal infections is significant Secondly, recent studies haveshown that mothers with periodontitis are at greater risk for having lowweight babies than those without periodontitis (Offenbacher et al., J.Periodontol., 67:1103 (1996)).

There is also growing acceptance that diabetes is associated withincreased occurrence and progression of periodontitis-so much so thatperiodontitis has been called the “sixth complication of diabetes” (Loe,Diabetes Care, 16(1): 329-34 (1993)). The risk is independent of whetherthe diabetes is type 1 or type 2. Type 1 diabetes is the condition inwhich the pancreas produces little or no insulin. It usually begins inchildhood or adolescence. In type 2 diabetes, secretion and utilizationof insulin are impaired; onset is typically after age 30. Together,these two types of diabetes affect an estimated 15.7 million people inthe United States and represent the seventh leading cause of death(National Institute of Diabetes and Digestive and Kidney Diseases(NIDDK). Diabetes statistics. NIH Pub. No. 99-3892 (1999)). The goal ofdiabetic care is to lower blood glucose levels to recommended levels.Some investigators have reported a two-way connection between diabetesand periodontal disease, proposing that not only are diabetic patientsmore susceptible to periodontal disease, but the presence of periodontaldisease affects glycemic control.

D. Oral Disease and Adverse Pregnancy Outcomes

Preterm birth and low birth weight are considered the leading perinatalproblems in the United States (Gibbs et al., Am. J. Obstet. Gynecol.,166(5): 1515-28 (1992)). Although infant mortality rates have decreasedsubstantially over the past generation, the incidence of low birthweight (just under 300,000 cases in 1995) has not shown a comparabledecline (Institute of Medicine, Committee to Study the Prevention of LowBirth Weight, Division of Health. Promotion and Disease Progression.Preventing low birth weight. Washington: National Academy Press (1985)).Over 60 percent of the mortality of infants without structural orchromosomal congenital defects may be attributed to low birth weight(Shapiro, et al., Am. J. Obstet. Gynecol., 136(3): 363-73 (1980)).

Oral disease may contribute to adverse outcomes of pregnancy as aconsequence of a chronic oral inflammatory bacterial infection. Forexample, toxins or other products generated by periodontal bacteria inthe mother can reach the general circulation, cross the placenta, andharm the fetus. In addition, the response of the maternal immune systemto the infection elicits the continued release of inflammatorymediators, growth factors, and other potent cytokines, which maydirectly or indirectly interfere with fetal growth and delivery.

Evidence of increased rates of amniotic fluid infection, chorioamnioninfection, and histologic chorioamnionitis supports an associationbetween preterm birth, low birth weight, and general infection duringpregnancy. It is noteworthy that the largest proportion of suchinfections occurred during the pregnancies of the most premature births(Hillier, et al., N. Engl. J. Med. 319(15): 972-8 (1988); Hillier, etal., N. Engl. J. Med., 333(26):1737-42 (1995)). The biologicalmechanisms involve bacteria-induced activation of cell-mediated immunityleading to cytokine production and the synthesis and release ofprostaglandins, which may trigger preterm labor (Hillier, et al.,supra). Elevated levels of prostaglandin as well ascytokines-(interleukin-1 (IL-1), interleukin-6 (IL-6), and tumornecrosis factor alpha (TNF-α) have been found in the amniotic fluid ofpatients in preterm labor with amniotic fluid infection (Romero, et al.,Am. J. Obstet. Gynecol., 186(6 Pt 1):1654-64 (1993)), compared withlevels in patients with preterm labor without infection.

E. Mucosal Infections

The mucosal lining of the mouth is subject to a variety of infectionsand conditions, ranging from benign canker sores to often fatal cancers.

Oral Candidiasis

Chronic hyperplastic candidiasis is a red or white lesion that may beflat or slightly elevated and may adhere to soft or hard tissuesurfaces, including dental appliances. It is caused by species ofCandida, especially Candida albicans, the most common fungal pathogenisolated from the oral cavity. Normally, the fungi are present inrelatively low numbers in up to 65 percent of healthy children andadults and cause no harm (McCullough, et al., Int. J. Oral Maxillofac.Surg., 25:136-44 (1996)).

The most common form of oral candidiasis is denture stomatitis. Itoccurs when tissues are traumatized by continued wearing of ill-fittingor inadequately cleaned dental appliances and is described as chronicerythematous candidiasis. Another form, candidal angular cheilosis,occurs in the folds at the angles of the mouth and is closely associatedwith denture sore mouth (Tyldesley and Field, Oral Medicine, 4th ed.,Oxford University Press (1995)). Other common forms of Candida infectionare pseudomembranous candidiasis (thrush), which may affect any of themucosal surfaces, and acute erythematous candidiasis, a red and markedlypainful variant commonly seen in AIDS patients.

In most cases, Candida infection may be controlled with antifungalmedications used locally or systemically. Control is difficult, however,in patients with immune dysfunction, as in AIDS, or other chronicdebilitating diseases. Often the organisms become resistant to standardtherapy, and aggressive approaches are necessary (Tyldesley and Field,supra). The spread of oral candidiasis to the esophagus or lungs may belife-threatening and is one of the criteria used to define frank AIDS(Samaranayake and Holmstrup, J. Oral Pathol. Medi., 18:554-64 (1989)).

F. Herpes Simplex Virus Infections

In any given year, about one-half-million Americans will experiencetheir first encounter with the herpes simplex virus type 1 (HSV-1), thecause of cold sores. That first encounter usually occurs in the oralregion and can be so mild as to go unnoticed. But in some people,particularly young children and young adults, infection may take theform of primary herpetic stomatitis, with symptoms of malaise, muscleaches, sore throat, and enlarged and tender lymph nodes, prior to theappearance of the familiar cold sore blisters. These blisters usuallyshow up on the lips, but any of the mucosal surfaces may be affected.Bright-red ulcerated areas and marked gingivitis can also be seen(Tyldesley and Field, supra).

Herpes viruses also cause genital infections, which are transmittedsexually. Both HSV-1 and HSV-2 have been found in oral and genitalinfections, with HSV-1 predominating in oral areas and HSV-2 in genitalareas (Wheeler, J. Am. Acad. Dermatol., 18(1 Pt. 2): 163-8 (1988)).Herpes viruses have also been implicated as cofactors in the developmentof oral cancers. Crowded living conditions may result in greater contactwith infected individuals, which aids in transmission of HSV (Whitley,Pathol. Biol., 40(7): 729-34 (1992)).

G. Oral Human Papillomavirus Infections.

There are more than 100 recognized strains of oral human papillomavirus(HPV), a member of the papovavirus family, implicated in a variety oforal lesions (Regezi and Sciubba, Oral pathology. Clinical-pathologiccorrelations, 2nd ed., (1993)). Most common are papillomas (warts) foundon or around the lips and in the mouth. HPV is found in 80 percent ofthese oral squamous papillomas (de Villiers, Biomed Pharmacother.,43:31-6 (1989)). The virus has also been identified in 30 to 40 percentof oral squamous cell carcinomas (Chang, et al., Arch. Dermatol. Res.,282(8):493-497 (1990)) and has been implicated in cervical cancer aswell. Whether a cancer or nonmalignant wart develops may depend on whichvirus is present or on which viral genes are activated. Oral warts aremost often found in children, probably as a result of chewing warts onthe hands. In adults, sexual transmission from the anogenital region mayoccur (Franchesi, et al., Cancer Epidemiol. Biomarkers Prev., 5:565-575(1996)). In general, viral warts spontaneously regress after 1 or 2years. The immune system normally keeps HPV infections under control, asevidenced by the increased prevalence of HPV-associated lesions inHIV-infected patients and others with immunodeficiency.

H. Recurrent Aphthous Ulcers

Recurrent aphthous ulcers (RAU), also referred to as recurrent aphthousstomatitis, is the technical term for canker sores, the most common andgenerally mild oral mucosal disease. Between five and twenty-fivepercent of the general population is affected, with higher numbers inselected groups, such as health professional students (Ferguson, et al,J. Oral Med., 39(4):212-217 (1984); Kleinman, et al., Community Dent.Oral Epidemiol., 5:140-144 (1991)).

The disease takes three clinical forms: RAU minor, RAU major, andherpetiform RAU. The minor form accounts for 70 to 87 percent of cases.The sores are small, discrete, shallow ulcers surrounded by a red haloappearing at the front of the mouth or the tongue. The ulcers, whichusually last up to two weeks, are painful and can make eating orspeaking difficult. About half of RAU patients experience recurrencesevery one to three months; as many as thirty percent report continuousrecurrences (Bagan et al., J. Oral Pathol. Med., 20:395-7 (1991)).

RAU major accounts for seven to twenty percent of cases and usuallyappears as one to ten larger coalescent ulcers at a time, which maypersist for weeks or months (Bagan, et al., supra). Herpetiform RAU hasbeen reported as occurring in seven to ten percent of RAU cases. Theulcers appear in crops of ten to one hundred at a time, concentrating inthe back of the mouth and lasting for seven to fourteen days (Bagan, etal., supra).

RAU can begin in childhood, but the peak period for onset is the seconddecade (Lehner, Proc. R. Soc. Med., 61:515-24 (1968)). About fiftypercent of close relatives of patients with RAU also have the condition(Ship, J. Dent. Res., 44:837-44 (1965)), and a high correlation of RAUhas been noted in identical but not fraternal twins. Associations havebeen found between RAU and specific genetic markers (Scully and Porter,J. Oral Pathol. Med., 18:21-7 (1989)).

RAU has also been associated with hypersensitivities to some foods, fooddyes, and food preservatives (Woo and Sonis, J. Am. Dent. Assoc.,127(8):1202-13 (1996)). Nutritional deficiencies-especially in iron,folic acid, various B vitamins, or combinations thereof-have also beenreported, and improvements noted with suitable dietary supplements(Nolan, et al., J. Oral Pathol. Med., 20:389-91 (1991)).

I. Oral and Pharyngeal Cancers and Precancerous Lesions

Oral cancer is the sixth most common cancer in U.S. males and takes adisproportionate toll on minorities; it now ranks as the fourth mostcommon cancer among African American men (Kosary, et al., SEER CancerStatistics Review, NIH Pub. No. 96-2789 (1995)). The most common oralsites are on the tongue, the lips, and the floor of the mouth.

Viruses that have been implicated in oral cancer include herpes simplextype 1 and human papillomavirus. Epstein-Barr virus, also a herpesvirus, is now accepted as an oncogenic virus responsible for Burkitt'slymphoma, occurring primarily in Africa, and nasopharyngeal carcinoma,occurring primarily in China. HPV is a major etiologic agent in cervicalcancer, and has been found in association with oral cancer as well(Sugerman and Shillitoe, Oral Dis., 3:130-47.(1997)). HPV DNA sequenceshave been found in oral precancerous lesions as well as in squamous cellcarcinomas (Syrjanen, et al., J. Oral Pathol., 17(6):273-8 (1988)), andexperimental evidence has shown that HPV-16 may be an important cofactorin oral carcinogenesis (Park et al., Oncogene, 10(11: 2145-53 (1995)).Herpes simplex type 1 antibodies were demonstrated in patients with oralcancer, and herpes was found to induce dysplasia (abnormal cellularchanges) in the lips of hamsters when combined with the application oftobacco tar condensate.

More recently, human herpes virus 8, a newly identified member of theherpes virus family, has been found in Kaposi's sarcoma, an otherwiserare cancer occurring in patients with AIDS. These tumors often appearinitially within the oral cavity (Epstein and Scully, Int. J. OralMaxillofac. Surg., 21(4):219-26 (1992)). Other uncommon oral malignanttumors, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma, may alsooccur in the mouths of AIDS patients. In addition to viruses, infectionwith strains of the fungus Candida albicans has been linked to thedevelopment of oral cancers via the fungal production of nitrosamines,which are known carcinogens.

J. Associated Autoimmune Disorders

Oral, dental, or craniofacial signs and symptoms play a critical role inautoimmune disorders such as Sjogren's syndrome, and in a number ofchronic and disabling pain conditions. Sjogren's syndrome is one ofseveral autoimmune disorders in which the body's own cells and tissuesare mistakenly targeted for destruction by the immune system. Like otherautoimmune conditions, Sjogren's syndrome is more prevalent among women.The ratio of females to males affected is 9:1, with symptoms usuallydeveloping in middle age. There are an estimated one to two millionindividuals in the. United States with Sjogren's syndrome (Talal, Rheum.Dis. Clin. North Am., 18(3):507-15 (1992)).

The disease occurs in two forms. Primary Sjogren's involves the salivaryand lacrimal (tear) glands. In secondary Sjogren's the glandularinvolvement is accompanied by the development of a connective tissue orcollagen disease, most often rheumatoid arthritis, lupus erythematosis,scleroderma, or biliary cirrhosis.

The glandular involvement causes a marked reduction in fluid secretion,resulting in xerostomia and xerophthalmia (dry eyes). The constant oraldryness causes difficulty in speaking, chewing, and swallowing; the dryeyes often itch and feel gritty. There is no cure for Sjogren's, andpatients often carry eye drops and water bottles or saliva substitutesin an attempt to provide symptomatic relief. Clinically, the reductionin salivary flow changes the bacterial flora, which, in addition to thereduction in salivary protective components, increases the risk ofcaries and candidiasis (Daniels and Fox, Rheum. Dis. Clin. North Am.,18:571-589 (1992)). Recent studies have indicated that there is areduction in masticatory function (Dusek, et al. Gerodontology 13(1):3-6 (1996)) and an increased prevalence of periodontal disease (Najera,et al., Oral Surg. Oral Med. Oral Pathol Oral Radiol. Endod.,83(4):453-7 (1997)). In advanced stages the salivary glands can swellbecause of obstruction and infection or lymphatic infiltration. In bothforms of the disease, other systems can eventually become affected.Nasal, laryngeal, and vaginal dryness can occur, as well asabnormalities in internal organs (Oxholm and Asmussen, J. Inter Med,239:467-474 (1996)). Patients with Sjogren's syndrome are at some riskof developing diseases such as non-Hodgkin's lymphoma; clinical dataindicate that such lymphomas develop in 5 percent of patients withSjogren's syndrome (Moutsopoulos et al., Am. J. Med., 64(5):732-741(1978)).

K. HIV and Osteoporosis

The mouth may serve as an early warning system, diagnostic of systemicinfectious disease and predictive of its progression, such as with HIVinfection. In the case where oral cells and tissues have counterparts inother parts of the body, oral changes may indicate a common pathologicalprocess. During routine oral examinations and perhaps in futurescreening tests, radiographic or magnetic resonance imaging of oral bonemay be diagnostic of early osteoporotic changes in the skeleton.

L. HIV Infection

The progressive destruction of the body's immune system by HIV leads toa number of oral lesions, such as oral candidiasis and oral hairyleukoplakia, that have been used not only in diagnosis but also indetermining specific stages of HIV infection. Oral candidiasis is rarelyseen in previously healthy young adults who have not received priormedical therapy such as cancer chemotherapy or treatment with otherimmunosuppressive drugs. Oral candidiasis may be the first sign of HIVinfection and often occurs as part of the initial phase of infection—theacute HIV syndrome (Tindall, et al., “Primary HIV infection: Clinical,Immunologic, and Serologic Aspects,” in The Medical Management of AIDS,Sande, et al., eds., pp. 105-129; W. B. Saunders, 1995). It tends toincrease in prevalence with progression of HIV infection when CD4lymphocyte counts fall. It also appears to be the most common oralmanifestation in pediatric HIV infection (Kline, Pediatrics,97(3):380-388 (1996) and has been demonstrated to proceed to esophagealcandidiasis, a sign of overt AIDS. (Saah et al., Am. J. Epidemiol.,135:1147-1155 (1992)). Both the pseudomembranous and the erythematousforms of candidiasis appear to be important predictors of progression ofHIV infection (Klein et al., AIDS, 6(3): 332-333 (1992)).

Like oral candidiasis, oral hairy leukoplakia in HIV-positive personsheralds more rapid progression to AIDS. Oral hairy leukoplakia is anoral lesion first reported in the early days of the AIDS epidemic. Sinceits discovery, hairy leukoplakia has been found in HIV-negative personswith other forms of immunosuppression, such as organ or bone marrowrecipients and those on long-term steroid therapy, and less frequentlyamong immunocompetent persons.

Linear gingival erythema and necrotizing ulcerative periodontitis may bepredictive of progression of HIV infection. (Mealey, Ann. Periodontaol.,1:256-321 (1996)). Necrotizing ulcerative periodontitis, a more seriousperiodontal condition observed in HV-infected persons, is a goodpredictor of CD4+ cell counts of under 200 per cubic millimeter. Inaddition, the numerous ulcerative and nonulcerative conditions thataffect the oral cavity may affect the biologic activity of HIV and areaffected by its treatments (Mealey, supra).

M. Osteoporosis and Oral Bone Loss

Osteoporosis, a degenerative disease characterized by the loss of bonemineral and associated structural changes, has long been suspected as arisk factor for oral bone loss. In addition, measures of oral bone losshave been proposed as potential screening tests for osteoporosis.Osteoporosis affects over 20 million people in the United States, mostof whom are women, and results in nearly 2 million fractures per year(National Institute of Arthritis, Musculoskeletal and Skin Diseases2000). The disease is more prevalent in white and Asian American womenthan in black women. Oral bone loss has been reported to be moreprevalent in women than in men. Also, the association between estrogenstatus, alveolar bone density, and history of periodontitis inpostmenopausal women has been studied (Payne et al., J. Periodontol.,6:24-31 (1997)).

Larger cross-sectional studies, as well as longitudinal and mechanismstudies, are needed to better define the relationship betweenosteoporosis, osteopenia, and oral bone loss, periodontal disease, andtooth loss. The role of factors involved in the regulation of bonemineral density in men as well as in postmenopausal women needs to beevaluated further with reference to oral bone loss, tooth loss, andperiodontal disease. Variables such as sex, race, dietary calcium andphosphorus, vitamin D intake, exercise, body mass index, smoking,genetics, medication use, reproductive history, and psychosocial factorsneed to be assessed in depth. In addition, reliable and valid criteriaand imaging technologies for assessing osteoporosis and oral bone lossare needed to better elucidate the full relationship between skeletaland mandibular bone mineral density, periodontal disease, alveolar ridgeresorption, and tooth loss.

II. Salivary Mucins

The functional properties of saliva proteins, known as salivary mucins,relative to oral health status are the subject of continuing research(Ayad, et al., J. Dent. Res., 79:976-982 (2000)). The existence ofhigh-molecular-weight glycoproteins in saliva and saliva secretions,called mucins, has been recognized for nearly thirty years (Offner, etal., Adv. Dent. Res., 14:69-75 (2000)). Mucins are essential for oralhealth and perform many diverse functions in the oral cavity. Forexample, mucins are the principal protein components of the mucous layerwhich coats epithelial surfaces in the gastrointestinal, respiratory,and reproductive tracts. This layer forms a viscous barrier whichprotects the underlying epithelium from desiccation, mechanical injury,and microbial assault, while allowing for active absorption andsecretion by mucosal cells. Mucins are also secreted by salivary glandsand are thought to have a major role in the protection of oralepithelial surfaces, as well as in the non-immune host defense system inthe oral cavity (Offner, et al., supra).

From a biochemical standpoint, mucins are comprised of approximately15%-20% protein, and up to 80% carbohydrate, present largely in the formof O-linked glycans (Strous and Dekker, Crit. Rev. Biochem. Mol. Biol.,27:57-92 (1992); Gendler and Spicer, Ann. Rev. Physiol., 57:607-634(1995)). Serine and threonine are the most abundant amino acids andserve as the attachment sites for these carbohydrate chains. Many mucinshave monomeric molecular weights greater than two million Daltons, andform multimers more than ten times that size (Offner et al., supra). Todate, eleven distinct human mucin genes have been isolated and have beennumbered MUC1-MUC4, MUC5AC, MUC5B, MUC6-8, and MUC11-MUC12, in the orderof their discovery.

These mucins share several common properties. The polypeptide backbonecan be divided into three regions. The central region is enriched inserine, threonine, and sometimes proline, and contains tandemly repeatedsequences ranging in length from 8 to 169 amino acids. This domainserves as the attachment site for the O-glycans, and each mucin has aunique, signature tandem-repeat sequence. The N— and C-terminal regionsof mucins are non- or sparsely glycosylated with both O— and N-linkedsugars. In many mucins, these flanking regions are cysteine-rich,containing nearly 10% cysteine. Mucins could be organized into threedistinct classes: the large gel-forming mucins (i.e., MUC2, MUC5AC,MUC5B, and MUC6); the large membrane-associated mucins (i.e., MUC1,MUC3, MUC4, and MUC12); and the small soluble mucins represented byMUC7. Insufficient information is available to assign MUC8 and MUC 11 toone of these categories (Ofnner, et al., supra).

The MUC7 gene has previously been reported (Bobek, et al., Genomics,31:277-282 (1998)). The MUC7 mucin is generally regarded as having theability to bind to and aggregate several species of oral bacteria,including several strains of S. mutans, and A. actinomycetemcomitans.The former is thought to be the most cariogenic of the oral bacteria andthe latter is one of two major pathogens in periodontal disease. The.MUC7 mucin also binds C. albicans and can have candidicidal activity.Desialylation of the mucin apparently destroys its ability to aggregatesome species of oral bacteria. Recent studies further indicate that MUC7mucin binds oral neutrophils on a different oligosaccharide motif thanis used to bind oral bacteria. With regard to the primary site ofbinding to oral bacteria, recent studies suggest that a non-glycosylateddomain of MUC7 mucin can be more responsible than its oliogosaccharides.

Bolscher et al., (J. Dent. Res., 78:1362-1369 (1999)) have developed anELISA for MUC7 that quantitated mucin in stimulated separate salivasfrom the parotid, submandibular, and sublingual glands of six subjects.The Bolscher study reported the mean concentration of MUC7 for each ofthe three glandular salivas. Rayment et al. (J. Dent. Res., (2000)19:1765-1772) have developed a capture ELISA for MUC7 and measured themean concentration in the stimulated whole saliva of 61 subjects.

In addition, others have either studied the functional properties ofMUC7 or the concentrations of other saliva proteins relative to oralhealth status. Prakobphol, et al. have studied the different types ofoligosaccharide chains on MUC7 (Biochemistry 38:6817-6825 (1999)).Prakobphol, et al. reported that different individuals have differentclasses of oligosaccharides on their MUC7, but they did not include apopulation study, nor did they quantitate the mucin or itsoligosaccharides. Bobek, et al. first cloned the MUC7 gene and is nowstudying its functional properties (Genomics 31:277-282 (1998)). Ayad,et al., have systematically tested the relationship of variouscomponents in saliva to oral health status (J. Dent. Res. 79:976-982(2000)).

U.S. Patent Publication No. 20003/0040009 A1 to Denny et al., which isincorporated herein by reference, describes the relationship of mucinconcentration to DFT (decayed and filled permanent teeth). The mucintest, as described in U.S. Patent Publication No. 20003/0040009 A1,comprises first separating a salivary mucin, e.g., MUC7 mucin, from allother sialic acid-containing molecules in the saliva, by known methodssuch as sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The sialic acid attached to the mucin is then quantitatedand reported.

There have been many attempts to measure other factors in saliva andthen relate such factors to the caries experience of the individualdonors. However, the vast majority of studies, which have reportedvalues for pH, various ions, macromolecules, and flow rate, have foundlittle evidence of a correlation. A few studies have shown a smallamount of correlation, though without consensus between studies. Thus itwould be of value to develop a test that exhibits a strong correlationbetween a specific factor and the risk level for caries development, andwould allow for the prediction or forecasting of existing caries levels.In addition, this test should be simple and accurate.

Despite advances, caries remains a disease experienced by 85% of U.S.school children before the age of 17. Though wide spread, approximately75% of all caries is concentrated in only 25% of the population (U.S.Dept. of Health & Human Services. National Institutes of HealthConsensus Development Conference Statement. Diagnosis and Management ofDental Caries Throughout Life (2001)). At present, these caries-proneindividuals are identified by accumulated caries experience, usually inan office setting. However, this professional judgment is based onpersonal anecdotal experience. Literature on the prediction of futurecaries development in specific individual subjects is lacking. Thecurrent level of scientific effort is reflected in 24 studies of theefficacy of caries preventives and non-invasive treatments, whosesubject populations were selected on -the basis of “high-risk” or“caries-active.” These studies are summarized in “EvidenceReport/Technology Assessment, Number 36: Diagnosis and Management ofDental Caries” (U.S. Dept. of Health & Human Services. NationalInstitutes of Health Consensus Development Conference StatementDiagnosis and Management of Dental Caries Throughout Life (2001)). Withaverage ages ranging from 1 to 13 years, these studies averaged 175individuals per study. Each study was evaluated on the quality of theevidence for judging the efficacy of a particular treatment. The fourgrades achievable were “good,” “fair,” “poor,” and “incomplete.” Onlyfour of the individual studies received a fair rating for their resultsand conclusions. The remaining 20 were rated as incomplete. While anumber of factors were at play in this rating, special note was made ofthe lack of consistency in the inclusion criteria for “at-risk” and“caries-active” participants.

In general, these studies have used either Streptococcus mutans titersor past caries history, such as dmft or dmfs (deciduous teeth), and DMFTor DMFS (permanent teeth), to select the high risk and/or caries-activesubjects. A problem associated with these inclusion criteria is thesubjectiveness associated with the-actual diagnoses, as well as lack ofagreement on what number constitutes a caries-prone individual. Thisuncertainty about identification of individuals who have a highpotential for future caries development is exemplified in a dividedstudy that was partitioned into “high-risk” (S. mutans titer-based), and“caries-active” (DMFS-based) subjects. The same treatment agent gave a33% reduction in new caries in the “high-risk” group and a 9% increasein the “caries-active” group (U.S. Dept. of Health & Human Services.National Institutes of Health Consensus Development ConferenceStatement. Diagnosis and Management of Dental Caries Throughout Life(2001)). In addition to the confusion about what inclusion criteria bestidentify those with the highest projected rates of cariogenesis, theassessment report notes that neither approach to caries risk assessmenthas ever been validated.

Thus, there is value and need for a test that provides standardsubject-identification criteria and that leads to a consistent diagnosisthroughout childhood and early adulthood. A preferred test would have auniform design such that it would accommodate all age groups and races.

SUMMARY OF THE INVENTION

This present invention provides methods and test devices for predicting,assessing, and diagnosing the risk of a disease using salivary analysis.It is not intended that the present invention be limited to compositionsand methods for predicting and preventing specific diseases. Thus, thepresent invention provides methods for predicting and reducing the riskof a disease and diagnostic kits for detecting a disease based onmeasurement of the content of lectin-binding components in a whole(i.e., unfractionated) saliva sample.

More specifically, one aspect of the present invention provides a methodfor predicting the risk of a disease in a subject, comprising providingan unfractionated saliva sample from a subject; contacting an aliquot ofthe saliva with one or more lectins under conditions that allow the oneor more lectins to bind to lectin-binding components of the saliva;detecting the amount of bound lectin; and comparing the amount of boundlectin to the amount known to bind a saliva sample from a controlpatient (i.e., a patient or patients without the disease), wherein theamount detected is indicative of the risk of the disease. The salivasample may be a stimulated saliva sample or, in a preferred embodiment,an unstimulated saliva sample. The lectin-binding components may be anyof the salivary oligosaccharides that bind lectins. In one embodiment,the lectin is MAL I. In another embodiment, the lectin is selected froma lectin other than PNA.

In one embodiment, the contacting and detecting steps are part of aWestern blot procedure. For example, in one embodiment, the contactingcomprises applying a drop of said saliva sample to a matrix material,and then contacting the matrix material with a solution of said one ormore lectins. In another embodiment, the contacting comprises applyingone or more lectins to said matrix material; and then contacting thematrix-bound lectins with the saliva sample.

A further aspect of this invention provides a method of using one ormore lectins for predicting the risk of a disease, comprising providingan unfractionated saliva sample from a subject; providing one or morelectins that bind to one or more oligosaccharide components of thesaliva; contacting the saliva sample with the one or more lectins underconditions that allow the one or more oligosaccharide components to bindto the one or more lectins; and detecting the amount of bound lectins,wherein the amount of bound lectins correlates with the risk of thedisease. In one embodiment, the contacting and detecting steps are partof a Western blot procedure.

In one embodiment, the methods of the present invention can furthercomprise the step of assessing the risk of the disease at a definedlevel, such as high, medium, low, very low or zero. In addition, themethods of the present invention can further comprise the step ofassessing the risk of future development of a disease in a subject.

Another aspect of this invention provides a method for preventing orreducing the risk of a disease, comprising providing an unfractionatedsaliva sample from a subject; contacting an aliquot of the saliva withone or more lectins under conditions that allow the one or more lectinsto bind to a lectin-binding component of the saliva; detecting theamount of bound lectin; comparing the amount of bound lectin to theamount known to bind a saliva sample from a control subject, wherein theamount is proportional to the risk of a disease in said subject; andadministering a therapeutic reagent to the subject when the content ofthe component in the saliva is above or below the level expressed innormal control (i.e., a subject free from the disease being tested for).The oral fluid standard can comprise a sample from a control (i.e., asubject who does not suffer from the disease being tested for).

Another aspect of this invention provides a diagnostic kit for detectinga disease, comprising: a) a means for collecting a saliva sample; b) ameans for measuring the amount of a lectin-binding component in thesample; and c) an oral fluid standard for comparing the amount of thecomponent in the sample. In one embodiment of the present invention, thekit is a Western blot format. In kits of this nature, a drop of a salivasample is transferred onto a matrix material (e.g., a nitrocellulosefilter). The drop is then probed with one or more lectins, and thelectins are detected by means of suitable methods.

It is not intended that the methods and devices of the present inventionbe limited to predicting diseases of human subjects within a particularage group or race. Preferably, the methods and devices of this inventionare universal in that the accommodate subjects of all ages and races.

The diseases that can be predicted according to the methods of thisinvention include, but are not limited to, oral diseases and associatedmedical disorders. Oral diseases and associated medical disordersinclude, but are not limited to, dental caries; periodontal diseases(e.g., gingivitis, adult periodontitis, early-onset periodontitis,etc.); diseases associated with periodontal disorders (e.g., pulmonaryand respiratory diseases, and cardiovascular diseases such as heartattack, stroke, atherosclerosis, etc.); diabetes; perinatal disorders(e.g., low birth weight and premature births); mucosal infections; oraland pharyngeal cancers; precancerous lesions; associated autoimmunedisorders (e.g., Sjorgren's syndrome); HIV; and osteoporosis.

In preferred embodiments, the present invention provides a method forpredicting dental caries risk. The dental caries can be early-onsetdental caries, adult dental caries, root caries, DFT, DMF, DFS or DMFSin children and adults. In children dental caries can also include dft,dmf, dfs, dfs/t or dmfs.

Additional advantages and novel features of this invention shall be setforth in part in the description that follows, and in part will becomeapparent to those skilled in the art upon examination of the followingspecification or may be learned by the practice of the invention. Theadvantages of the invention may be realized and attained by means of theinstrumentalities, combinations, compositions, and methods particularlypointed out in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

This patent contains at least one drawing executed in color. Copies ofthis patent with colored drawings will be provided by the office uponrequest and payment of the necessary fee.

FIG. 1 is an image of a dot blot test of saliva samples from twoindividuals according to this invention using a lectin panel comprisingJacalin, SNA, ACL, AAL, UEA, and MAL I.

FIG. 2 describes a linear regression anaylsis of DFT versus the sum ofindependent variables (MAL I, JAC, MAA, MUC7 mucin, MUC5B mucin, gender,and age) for forecasting DFT with representative 98% confidence levels.

FIG. 3 describes a linear regression analyses for the relationshipbetween MUC7 mucin alone and DFT with representative 98% confidencelevels for the same subjects analyzed in FIG. 2.

FIG. 4 is a dot blot of mouse sublingual mucin probed with specificantibody-secondary antibody HRP.

FIG. 5 is a dot blot of mouse sublingual mucin probed with specificantibody-secondary antibody-AP.

FIG. 6 is a dot blot of whole saliva from children with Low and High DFTfollowing MAL I and AMCA incubations and long wave UV.

FIG. 7 describes a linear regression analyses of DFT versus the sum ofindependent variables (MAL I, JAC, SNA, ethnicity, and age) forforecasting DFT with representative 96% confidence levels.

FIG. 8 describes a linear regression analyses of high, medium, low, andzero risk versus the sum of independent variables (MAL I, JAC, SNA, andgender) for forecasting DFT with representative 96% confidence levels.

FIG. 9 is a Western blot assay of salivas from 8 subjects with thelectin MAL I.

FIG. 10 is a Western blot assay of salivas from 8 subjects with thelectin JAC.

FIG. 11 is a Western blot assay of salivas from 8 subjects with thelectin SNA.

FIG. 12 is a high throughput Western blot assay for SNA reactivity in0.2 and 0.1 μL of whole saliva from Griffin Study subjects.

FIG. 13 is a negative image of the Western blot shown in FIG. 12.

FIG. 14 is a graph showing the relationship of the results from a cariestest of this invention to each individual's accumulated caries history.DFS is the dependent variable in this plot.

FIG. 15 is a graph showing the relationship between risk level in youngadults versus the sum of quantitated lectin affinities.

FIG. 16 is a graph showing the relationship of dfs/t (number ofdeciduous caries per remaining deciduous teeth) and the sum ofquantitated lectin affinities.

FIG. 17 is a graph showing the relationship of the risk level fordeciduous caries in children and the sum of quantitated lectinaffinities.

FIG. 18 describes a linear regression analyses of DFT versus the sum ofquantitated lectin affinities with representative 96% confidence levelsin a mixed group of children and adults.

FIG. 19 describes a linear regression analyses of risk level forchildren and young adults versus the sum of quantitated lectinaffinities with representative 96% confidence levels.

FIG. 20 is a Western blot assay of salivas with lectins LTL and LELconjugated to blue and yellow colored microlatex beads, respectively.

FIG. 21 is a Western blot assay of salivas with lectins LTL and LELconjugated to red and yellow colored microlatex beads, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified and evaluated factors in saliva, i.e.,lectin-binding components of saliva, which may or may not be associatedwith mucins, that facilitate caries prediction, and have utilized thisdiscovery to develop a caries risk test that is suitable forcommercialization. These lectin-binding components are better and morebroadly applicable risk indicators than MUC7 and MUC5B mucins alone.Furthermore, it was discovered that for individuals whose mucin lacks alectin-binding oligosaccharide(s) or who lack mucin, the lectin-bindingoligosaccharides are present in quantity on other salivaryglycoproteins. Accordingly, the salivary analyses of this inventionprovide a more universal method for predicting and reducing the risk ofdisease. Thus, with the risk tests of this invention, groups of childrenand young adults for whom the mucins may not be forecasters ofaccumulated caries experience can still be identified with highprobability, even when grouped together with individuals for whom themucins are indicators. The lectin-binding components can be quantitatedwith less difficulty than the mucins alone and therefore provide simple,reliable tests.

More specifically, one aspect of this invention provides a method forpredicting the risk of a disease in a subject, comprising providing anunfractionated saliva sample from said subject; contacting an aliquot ofsaid saliva with one or more lectins under conditions that allow saidone or more lectins to bind to a component of said saliva; detecting theamount of bound lectin; and comparing the amount of bound lectin to theamount known to bind a saliva sample from a control patient or subject.In one embodiment, the disease is dental caries.

A “control” patient or subject as defined herein is a representativepatient or subject of known disease level defined, e.g., according tothe number of cavities. For example, in certain populations, 0-2 DFS(total Decayed and Filled tooth Surfaces in the permanent teeth) may beassociated with very low risk of developing caries in young adults,while 3-8 DFS may be associated with low risk, 9-16 DFS with mediumrisk, and 17 or more DFS with high risk (see Example 4). When multiplelectins are employed in a method of this invention, they may be usedeither individually or in groups. The amount of bound lectin may bedetermined (e.g., through direct visual observation with naked eyes) bycomparing it with one or more (i.e., a panel of) control amounts. Eachcontrol amount is the amount of bound lectin known to bind a salivasample from a control patient or subject. The risk of a disease is thendetermined based on the amount of bound lectin. For example, if theamount of bound lectin is comparable to that for the above-defined “verylow risk group,” the individual is predicted to be at risk of developing0-2 DFS.

An “adult” is defined according to National Institute of Healthstandards as a human that is at least 21 years old.

A “child” is defined according to National Institute of Health as ahuman that is less than 21 years old.

In one embodiment, the lectin-based caries risk tests of this invention,also referred to as “caries tests,” measure two outcomes: 1) cariesassessment and 2) risk evaluation. Caries assessment is described as theaccumulated caries history of an individual. Examples of metrics areDFS, (total Decayed and Filled tooth Surfaces in the permanent teeth,dfs, (total decayed and filled surfaces in deciduous (“baby”) teeth),and dfs/t, which refers to the number of decayed and filled surfaces perremaining deciduous teeth. Risk evaluation provides a projection of thenumber of caries an individual can expect to accumulate by a certain agein the absence of individualized preventive treatments. An example of ametric that is used according to this invention is the risk level scaleof high, medium, low, and very low. As will be seen these risk levelscan be linked directly to acquired ranges of DFT and dfs/t in the testpopulations. Risk may also be calibrated to more or less than fourlevels and can also be calibrated to different ranges of DFT or dft tobetter reflect the caries history patterns of different populations towhich the caries test may be applied.

The risk tests of this invention can be integrated into dental practiceas a common diagnostic procedure health screening, and in broader oralhealth campaigns to improve identification, treatment, and prevention inhigh-risk individuals. The diagnostic screening information provided bythese combinations of test and technology will aid the health careprovider in identifying caries-prone children, teenagers, and youngadults. This will be most helpful at stages of dental development whenphysical examination alone or dmfs (decayed, missing, and filledsurfaces) in deciduous teeth and DMFS in permanent teeth cannot identifywith statistical certainty, those individuals who are at-risk.

I. DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “saliva” refers to an oral fluid, regardless ofwhere the saliva is secreted in the oral cavity, or how it is collected.The saliva can be unstimulated or stimulated. In a preferred embodiment,the sample of saliva is unstimulated. As used herein, the term“unstimulated saliva” means that the subject will expectorate in acollection vessel without stimulation of salivary flow. For example, asubject's saliva may not be stimulated by chewing on a piece of paraffinfilm or tart candy. An “unfractionated” saliva sample means that none ofthe components of the saliva sample have been separated out of thesample that is to be used in a method or test of this invention.

As used herein, the terms “prediction of dental caries risk” or“prediction of dental caries experience” refer to the risk of futuredental caries development and the forecast of the current accumulatednumber of caries and fillings, respectively. Caries is a diseasecharacterized by demineralization of the dental enamel and of the dentinin various stages of progress, until it affects the pulp space. Fillingsrefer to those caries that have been treated or restored. “Prediction”is synonymous with the terms prognostication, forecasting, foretelling,foreseeing, portending, etc.

As used herein, the term “oral fluid standard” refers to a solutionuseful as a surrogate for naturally occurring oral fluid in the testing,calibration and standardization of oral fluid collection methods anddevices, oral fluid handling, preservation and storage methods anddevices, and oral fluid-based assay methods and devices. Oral fluidstandards are not intended as an in vivo therapeutic replacement orsupplement for saliva, but rather are used as ex vivo testing standards.The term oral fluid standard may refer to the oral fluid surrogatecomposition alone, or to the oral fluid surrogate spiked with one ormore additional components such as an analyte and/or human serum. Theparticular meaning of the term oral fluid standard will be apparent fromthe context in which it is used.

As used herein, the term “oral fluid” refers to one or more fluids foundin the oral cavity individually or in combination. Oral fluids include,but are not limited to saliva and mucosal transudate. It is recognizedthat oral fluids (e.g., saliva) are a combination of secretions from anumber of sources (e.g., parotid, submandibular, sublingual, accessoryglands, gingival mucosa and buccal mucosa), and the term oral fluidincludes the secretion of each of these sources individually or incombination.

As used herein, the term “mucins” refers to acid mucopolysaccharidescomplexed with proteins. The acid mucopolysaccharides are a group ofrelated heteropolysaccharides usually containing two types ofalternating monosaccharide units, of which at least one has an acidicgroup (typically either a carboxyl or a sulfuric group). The term “MUC7”refers to a particular mucin gene. The term “MUC7 mucin or occasionallyMUC7 protein” refers to the protein encoded by the MUC7 gene andpost-translationally modified to contain the necessary carbohydrates andpossibly sulfur to qualify it as a mucin, which is a recognizedbiochemical class of glycoproteins.

As used herein, the term “lectins” refers to proteins that bind, oftenwith great specificity, to defined oligosaccharide structures onglycoproteins and glycolipids.

As used herein, the term “subject” refers to a subject whose saliva isbeing tested for a particular disease. The subject can be a human or ananimal.

As used herein, the terms “normal subject” or “normal control” refer toa subject who does not suffer from the particular disease being testedfor (e.g., dental caries or any diseases associated with dental cariesexperience).

As used herein, the terms “oral disorders” and “oral diseases” refer todiseases and disorders affecting the oral cavity, and associated medicaldisorders. Oral disorders include, but are not limited to, dentalcaries; periodontal diseases (e.g., gingivitis, adult periodontitis,early-onset periodontitis, etc.); mucosal infections (e.g., oralcandidiasis, herpes simplex virus infections, oral human papillomavirusinfections, recurrent aphtous ulcers, etc.); oral and pharyngealcancers; and precancerous lesions.

As used herein, the term “associated medical disorders” refers tomedical conditions associated with periodontal diseases (e.g., pulmonaryand respiratory diseases, and cardiovascular diseases such as heartattack, stroke, atherosclerosis, etc.); associated autoimmune disorders(e.g., Sjorgren's syndrome); HIV; and osteoporosis.

A Western blot involves transfer of an aliquot (e.g., a drop) of asaliva sample to a membrane (e.g., nitrocellulose, nylon, or PVDF).Presence of a lectin-binding component in the saliva is then detected bycontacting the membrane with a solution containing one or more lectins,which are in turn detected by suitable means. Detection may be byautoradiography, calorimetric reaction or chemiluminescence. Forexample, the lectins can be conjugated to biotin, which in turn can bedetected and quantitated by contacting the bound lectin conjugates withfluorescently labeled avidin, or can be directly conjugated to anyreporter, including but not limited to microparticles such as microlatexbeads.

The present invention provides compositions and methods for assessingthe risk of a disease using salivary analysis. In particular, thecompositions and methods of the present invention can be used to predictand prevent the risk of oral diseases and other associated diseases.

II. SALIVARY ANALYSIS FOR PREDICTING DISEASES

The present invention provides new saliva-based methodologies andtechnologies for predicting the risk of and treating a disease.Specifically, the present invention relates to compositions and methodsfor predicting the risk of a disease based on analysis of salivarylectin-binding components. According to the invention, lectin-bindingcomponents of saliva, which may or may not be associated with mucins,can be probed with one or more lectins, and the lectin-binding activityof the saliva can be compared to the amount of lectin-binding activityof the saliva of a control subject, wherein the amount of bound lectinis indicative of the risk of the disease.

The lectin-binding components in saliva were discovered to be better andmore broadly applicable risk indicators than MUC7 and MUC5B mucinsalone. Furthermore, the present invention demonstrates that forindividuals in whom the mucin test does not apply, either their mucinlacks the specific lectin-binding oligosaccharide(s), or that lackingmucin, the oligosaccharide(s) are present in quantity on anothersalivary glycoprotein. Thus, according to one embodiment of theinvention, the risk of oral diseases and associated diseases ispredicted by quantitating the total lectin binding from unstimulated orstimulated saliva.

A. Lectins

Lectins are a family of proteins derived from a variety of plants,animals, and microbes. As a family they can be characterized by theirability to bind selectively to specific sugars or sugar linkages presentin the oligosaccharide chains of carbohydrate-bearing molecules. One ofthe most common uses of lectins is to ascertain individual blood types,of which there are at least 26 different families recognized at thistime. The A, B, O blood types represent only one of these families,which have as their common thread the types of sugar and linkages thatare represented in the carbohydrate-bearing molecules of an individual.As is commonly known, expression of blood types is genetically based.Some of the lectins used in the caries tests of this invention interactwith common blood types. All of the lectins that are used in the cariestests of this invention are commercially available.

The role of lectins in the caries tests of this invention is toquantitate a variety of types of sugars and their intermolecularlinkages associated with glycoproteins, glycolipids, complexpolysaccharides, and other carbohydrate-containing molecules present insaliva. The tests according to this invention are based on theintegration/combination of the quantitative results from a variety oflectins, some of which appear to be measuring carbohydrate propertiesthat are positively correlated with caries history and others that arenegatively associated with caries history. Individual outcomes of thecaries test represent the relative balance between positively andnegatively correlated lectin affinities in the saliva. The test may berun using a mixture of lectins or run with individual lectins whosequantitative results can then be mixed statistically.

In a preferred embodiment, the lectins used in the methods of thisinvention are one or more lectins selected from the group consisting ofDSL, ECL, PSA, WGA, UEA, MAL I, MAA, PNA, AAL, LTL, MAL II, JAC, LEL,SNA, PTL I, ACL, GSL II, VVA, BPL, WFL, SJA, MPL, GNL, HHL, CCA, NPL,STL, PHA-L, PHA-E, GSL I, DBA, HMA, EEA, LPA, and PTL II. In anotherembodiment, the lectins are selected from the group consisting of AAL,LTL and UEA 1. In yet another embodiment, the lectin is selected fromany lectin other than PNA.

In one nonlimiting example, a panel of lectins comprising DSL, ECL, PSA,MAL I, PNA, AAL, LTL, MAL II, JAC, LEL, PTL I, GSL II, VVA, BPL, SJA,MPL, and CCA can be used in a method of this invention to predict therisk for an adult for developing dental caries in permanent teeth.

In another nonlimiting example, a panel of lectins comprising ACL, PNA,LTL, PSA, MAL II, MAA, STL, PTL I, LEL, DSL, ECL, AAL, VVA, GNL I, CCA,SNA, JAC, WFL, SJA, MAL I, and BPL can be used in a method of thisinvention to predict the risk in a child for developing caries indeciduous teeth.

B. Sampling Methods

In a preferred embodiment, the methods of the present invention analyzean unstimulated or stimulated saliva sample to test for the risk of adisease. Saliva specimens for testing can be collected following variousmethods known in the art.

Proper conditions for generating unstimulated saliva have been described(Nazaresh and Christiansen, J. Dent. Res., 61:1158-1162 (1982)). Methodsand devices for collecting saliva have also been described (U.S. Pat.No. 5,910,122 to D'Angelo; U.S. Pat. No. 5,714,341 to Thieme et al.;U.S. Pat. Nos. 5,335,673 and 5,103,836 to Goldstein et al.; U.S. Pat.No. 5,268,148 to Seymour; and U.S. Pat. No. 4,768,238 to Kleinberg etal., each of which is incorporated herein in its entirety by reference).It is contemplated that the methods of the present invention can also bepracticed by analyzing stimulated saliva.

One preferred method of saliva collection utilizes a self-containedsterile device that is inserted into the mouth to collect a volume ofunstimulated saliva, which is then combined with an aliquot of includedsterile buffer. In an alternative embodiment, the sample is collected byhaving the subject lick or spit on a test strip of this invention.

Furthermore, the methods of the present invention are not limited toperforming salivary analysis immediately after collection of the sample.In other embodiments, salivary analysis following the methods of thepresent invention can be performed on a stored saliva sample. The salivasample for testing can be preserved using methods and apparatuses knownin the art (See, e.g., U.S. Pat. No. 5,968,746 to Schneider,incorporated herein its entirety by reference).

It is also contemplated that the methods of the present invention beused to perform salivary analysis on saliva samples that have beentreated to reduce its viscosity. Mucopolysaccharide-containing bodyfluids, such as saliva, contain antibodies and other metabolites thatare useful in the diagnosis of diseases, including those of bacterial,viral, and metabolic origin. However, the viscous nature of such fluids,due to the nature of mucopolysaccharides, makes testing of these fluidsdifficult. In order to prepare saliva for any laboratory testingprocedure, the saliva must be rendered sufficiently fluid (i.e.,viscosity must be reduced) and free from debris. Techniques used toremove debris include centrifugation and filtration. The viscosity ofsaliva can also be reduced by mixing a saliva sample with a cationicquaternary ammonium reagent (See, U.S. Pat. No. 5,112,758 to Fellman etal., incorporated herein in its entirety by reference).

Further, it is contemplated that the methods of the present invention beused in analyzing factors from saliva samples obtained from a subjectsuffering from xerostomia. Xerostomia is a condition in which thesalivary glands do not produce sufficient quantities of saliva. Theonset of the effects of xerostomia is insidious, with no clear line ofdemarcation when one suffers from the malady. It is estimated thatseveral million individuals suffer from this condition nationwide. Theactual number of individuals suffering from xerostomia is not known,however, because there has been little acknowledgement of the prevalenceor severity of the problem until recently. It is estimated that aboutten percent of the population over 50 years of age and 25 percent of thepopulation over 65 years of age suffer from xerostomia. The majority ofthose affected are women.

Some direct primary causes of xerostomia are autoimmune diseases, suchas Sjogren's syndrome, medical irradiation, malnutrition, hormonalimbalance, arthritis and aging. When areas of the head or neck aremedically irradiated by as little as 1000 rads per week, 85 percent ofthe patients suffer from xerostomia after six weeks and 95 percent afterthree months. Radiation xerostomia onsets rapidly, with a greater than50 percent decrease in salivary flow after one week, and a greater than75 percent decline after six weeks of treatment. The xerostomia isprogressive, persistent, and irreversible, reaching a greater than 95%reduction in saliva output three years after radiation. In patientswhere only part of the major salivary glands is in the path of theionizing radiation, the non-exposed portion can undergo hyperplasia andpartly compensate for the damaged acini. The most severe cases ofxerostomia are caused by radiation therapy after head and neck surgery,and by autoimmune diseases such as lupus, Sjogrens Syndrome, andrheumatoid arthritis (See, e.g., P. C. Fox, et al., J. Am. DentalAssoc., 110:519-525 (1985)). Secondarily, xerostomia is a side effectfrom the administration of over 400 drugs, including majorantihypertensives, antidepressants, antispasmodics, diuretics, musclerelaxants, antipsychotics, appetite depressants, and therapeutics forParkinson's disease.

To predict the risk of a disease in a subject suffering from xerostomia,it is contemplated that various methods for enhancing saliva be used toobtain a salivary sample for analysis. Various methods for enhancingsaliva are known in the art. For example, U.S. Pat. No. 5,886,054(incorporated herein in its entirety by reference) teaches a therapeuticmethod for enhancing saliva, using an aqueous solution of at least onepolymer and one electrolyte. The aqueous solution is preferably bufferedand optionally contains at least one mucin. In another example, U.S.Pat. No. 6,230,052 (incorporated herein in its entirety by reference)teaches an implantable device for inducing salivation by neuralstimulation at neurally sensitive location within an oral or perioraltissue of a user.

It is understood that the examples for sampling saliva described aboveare for illustrative purposes only. It is also understood that variousmodifications for sampling saliva are contemplated to be within thescope of the present invention.

C. Statistical Tests

In one embodiment, the methods of this invention for predicting the riskof a disease in a subject can further comprise the step of assessing therisk of the disease as high, medium, low, very low, or zero. In anotherembodiment, the methods of this invention for predicting the risk of adisease in a subject can further comprises assessing the risk of futuredevelopment of the disease in the subject. For example, the method ofassessing future risk can comprise comparing the amount of lectinbinding to a regression analysis derived from a group of subjectsexpressing a range of disease severity.

The statistical tests involved in the discovery of the predictive natureof lectin-binding components of saliva are standard and well-known inthe art of statistical analyses. Specifically, the present inventionused Pearson's correlation coefficient, simple linear regressionanalysis, multiple linear regression analysis, and ANOVA. An aspect ofthe reliability for the prediction of dental caries risk is the accuracyof representation involving the relationship between the test resultsand the observed decay or fillings on teeth (DFT) in the standardpopulation. In most statistical programs, this relationship iscalculated by the least squares method, and yields the mathematicalformula from which the linear regression line is derived and predictionscan be made. However, the typical regression line takes into accountonly the variation of the dependent variable. In a particularembodiment, the variation of dental caries experience is a dependentvariable.

In an embodiment of the invention where there is a normal variation inDFT and lectin-binding component concentration, a different type ofstatistical test can be used to give the most representativemathematical regression equation. This approach to regression analysiscan be performed by a variety of statistical tests, such as orthogonalleast squares, geometric mean regression, Bartlett's, three-group method(i.e., for Type II regression analysis), and random variable regressionanalysis. These alternative methods can also be used to calculate themathematical description of the regression line on the data. In thisembodiment, these methods did not measurably alter the predictiveoutcomes obtained by traditional simple linear regression analysis.

The R² of the regression analysis is preferably at least 0.3 (i.e., anynumber between and including 0.3 and 1). For example, for a 3-leveltest, the R² may be at least 0.9; for a 4-level test, the R² may be atleast 0.98; for a 5-level test, the R² may be at least 0.99.

D. Test Versions

In general, this invention provides a method for predicting the risk ofa disease in a subject, comprising providing an unfractionated salivasample from the subject; contacting an aliquot of the saliva with one ormore lectins under conditions that allow the lectin(s) to bind to alectin-binding component of the saliva; detecting the amount of boundlectin; and comparing the amount of bound lectin to the amount known tobind a saliva sample from a control subject, wherein the amount of boundlectin is indicative of the risk of the disease.

In one embodiment, the contacting and detecting steps are part of aWestern blot procedure. In one example, the procedure comprises applyinga drop of said saliva to a matrix material; and contacting the matrixwith a solution containing one or more lectins. In order to visualizethe amount of binding, the lectins can be coupled to a reporter or to acolored microparticle, as discussed below in detail.

Alternatively, the Western blot procedure comprises applying one or morelectins to a matrix material; and contacting the matrix-bound lectinswith the saliva sample.

The present invention also provides tests based on the methods of thisinvention for diagnosing diseases. In general, the test includes: i) ameans for collecting saliva; ii) a means for measuring the amount of oneor more lectin-binding components in the saliva; and iii) an oral fluidstandard for evaluating the amount of lectin-binding component.Different versions of the compositions and methods of the presentinvention can be used for various applications.

Preferably the test for predicting caries experience from saliva iseasily and accurately interpreted. In one embodiment, the presentinvention provides a practical test for predicting caries from a singlesaliva sample of young adults.

In a particular embodiment, the means for measuring the amount of one ormore lectin-binding components in a saliva sample comprises a strip testformat, analogous to a dot blot test. Preferably the strip test candistinguish multiple lectin-binding component concentrations. A striptest format provides various advantages over other possible designs inits ease of distribution, use, and interpretation.

Accordingly, one embodiment of an assay device comprises a test striphaving a sample receiving zone comprising a first matrix material forreceiving an aliquot of the saliva sample; and optionally a control zonecomprising a second matrix material having at least one controllectin-binding compound of a known concentration bound to the surface ofthe second matrix material. In one embodiment, a strip of this design ispackaged dry.

The test strip preferably comprises a semi-rigid support that has amatrix material laminated to one side of an end of the strip. Thesupport can be made of any suitable rigid or semi-rigid material, suchas poly(vinyl chloride), polypropylene, polyester, and polystyrene.Matrix materials suitable for purposes of this invention include, butare not limited to, nitrocellulose, cotton, polyester, rayon, nylon,polyethersulfone, and polyethylene.

The matrix material may be affixed to the support by any suitableadhesive means such as with a double-sided adhesive tape. Alternatively,the support may be a pressure sensitive adhesive laminate, e.g., apolyester support having an acrylic pressure sensitive adhesive on oneside that is optionally covered with a release liner prior toapplication to the matrix material.

Various ways of visualizing the amount of lectin that binds to thematrix-bound lectin-binding component can be used, and many methods areknown in the art, such as direct binding of specific visualizing stains(e.g., alcian blue, silver-enhanced alcian blue, or Stains-All);chromophore-labelled lectins; and various indirect methods, such asenzyme catalyzed amplification. The vast majority of the reagents to beused during development of the test are commercially available.

In accordance with one embodiment of the present invention, the lectinsmay be labeled with a reporter. As used herein, “reporter” refers to amoiety that provides the ability to detect a complex formed between thelectin and the lectin-binding component of the saliva. The reporter maybe detected by such characteristics as color change, luminescence,fluorescence, or radioactivity. Examples of reporters include, but arenot limited to, dyes, chemiluminescent compounds, enzymes, fluorescentcompounds, metal complexes, biotin, haptens, radioluminescent compounds,radioactive-labeled biomolecules, and colored microparticles. Oneskilled in the art can readily determine a suitable reporter once thetype of probe biopolymer to be utilized is determined.

In one embodiment, the labeling procedure may occur prior to analysis(direct labeling) or after complex formation (indirect labeling). Manybinding pairs are known in the art for indirect labeling, including, forexample, biotin-avidin, biotin-streptavidin, hapten-antihapten antibody,sugar-lectin, and the like. An example of indirect labeling would be thebiotinylation of a lectin, contacting the biotinylated lectin with themembrane-bound saliva sample, and reacting of the lectin/lectin-bindingcomponent complexes with a streptavidin-alkaline phosphatase conjugate.The lectin moieties that are retained after binding to thelectin-binding components in the saliva then bind to astreptavidin-alkaline phosphatase conjugate, which then acts on achromogenic substrate, such as Enzyme Labeled Fluorescent (ELF) reagent(Molecular Probes, Inc.).

According to one method of this invention, a test strip of thisinvention is spotted with a saliva sample and then contacted with asolution comprising a single lectin. In another embodiment, the spottedtest strip is contacted with a mixture of lectins. For example, thedesign of a test strip can be based on either spotting multipleconcentrations of the saliva sample (and therefore multipleconcentrations of the lectin-binding component) onto a immobilizingmatrix and a single intensity of color to be matched against a standard,or a single spot of the saliva sample and multiple intensities of colorsto be matched with a range of standard color intensities. The specificdesign will depend on the kinetics and affinities of various antibodyand dye/stain combinations.

For example, an assay device of this invention can comprise a test stripa sample having receiving zone on one portion the strip comprising afirst matrix material and one or more lectins bound to the first matrixmaterial; and optionally a control zone comprising a second matrixmaterial having at least one control saliva sample of a knownconcentration. In this embodiment, detection can comprise contacting thematrix material with a binding partner coupled to a reporter, whereinthe binding partner specifically binds the lectin-binding component.

An alternative method of visualizing and quantitating the amount oflectin binding involves the use of particles that can be directlyvisualized. That is, the lectin can be coupled to a particle, which canbe visualized in the test strip upon performing the assay. In anotherexample of this embodiment, the matrix spotted with the saliva samplecan be contacted with a mixture comprising a first lectin conjugated toa microparticle having a first color and a second lectin conjugated to amicroparticle having a second color, wherein the first and second colorsare distinguishable from one another. In one embodiment, the test isperformed to determine the risk of dental caries. In this embodiment,the first lectin is positively correlated with DFS and the second lectinis negatively correlated with DFS.

In an alternative embodiment, a method for predicting the risk of adisease in a subject comprises providing an unfractionated saliva samplefrom the subject; applying a drop of said saliva to a matrix material;contacting the matrix with a mixture of a first set of lectinsconjugated to a microparticle having a first color and a second set oflectins conjugated to a microparticle having a second color, whereinsaid first and second colors are distinguishable from one another;detecting the amount of bound lectins, preferably by visualization bythe naked eye; and comparing the amount of bound lectin to the amountknown to bind a saliva sample from a control subject, wherein the amountof bound lectin is indicative of the risk of the disease.

For example, each set of lectins may include one lectin or two or moredifferent lectins. Preferably, the first set of lectins is coupled tomicroparticles of a first color (e.g., blue) in proportions that reflecttheir contributions in the regression equation, and the second set ofdifferent lectins is coupled to microparticles of a second color (e.g.,yellow) that is distinguishable from the first color, also inproportions that reflect their contributions in the regression equation.The use of this mixture of two different colors of beads in an assay ofthis invention provides results in multiple colors on the test strip,e.g., ranging from blue, through the greens, and ending with yellow. Incertain cases, the yellow was difficult to see against a whitenitrocellulose matrix material. Thus, some red beads were added to theyellow at a ratio of 1:2 to make orange, which is much easier to see,but at that proportion still gives the green intermediate color withblue. Adding red and yellow in a ratio of 1:1 or greater gives brownintermediate colors rather than the green. In this example, the red andyellow beads are coupled to the same lectins.

The visible particles according to this invention are microparticles(i.e., a micrometer-sized particles) that can be directly visualized,such as a dyed particle. Any suitable insoluble particle may be employedfor purposes of this invention, including, but not limited to, particlesof a polymeric material which may include, but is not limited to, athermoplastic (e.g., one or more of polystyrenes, polyvinyl chloride,polyacrylate, nylon, substituted styrenes, polyamides, polycarbonate,polymethylacrylic acids, polyaldehydes, and the like), latex, acrylic,latex or other support materials such as silica, agarose, glass,polyacrylamides, polymethyl methacrylates, carboxylate modified latex,Sepharose, methacrylate, acrylonitrile, polybutadiene, metals, metaloxides and their derivatives, silicates, paramagnetic particles andcolloidal gold, dextran, cellulose, and liposomes, and natural particlessuch as red blood cells, pollens, and bacteria. The size of themicroparticles used in this invention is selected to optimize thebinding and detection of lectin-binding components of saliva, and aretypically 0.01 to 10.0 μm in diameter and preferably 0.01 to 1.0 μm indiameter, specifically not excluding the use of either larger or smallermicroparticles as appropriately determined. In one embodiment, themicroparticle is substantially spherical in shape. The preferredmicroparticle in the present invention is composed of latex containing acolored dye.

In accordance with the invention, the microparticles are coupled to orcomplexed with a lectin. Methods of coupling proteins such as lectins toparticles are well known in the art. For example, in one embodiment, themicroparticles possess surface sulfate charge groups that can bemodified by the introduction of functional groups such as hydroxyl,carboxyl, amine and carboxylate groups. The functional groups are usedto bind a wide variety of lectins to the microparticles, and areselected based on their ability to facilitate binding with the selectedlectin. Conjugation of the lectins to the microparticle is accomplishedby covalent binding or, in appropriate cases, by adsorption of thelectin onto the surface of the microparticle. Techniques for adsorptionor covalent binding of proteins to microparticles are well know in theart and require no further explanation.

The strip test format according to this invention is suitable for anumber of different applications, including, but not limited to:

1) A simple strip: this test could be used in a classroom setting toprovide a general expectation of caries experience (such as high,medium, low, and very low) for the dentally indigent student, as well asto assess the risk for future caries development. This version wouldalso be appropriate for use in underdeveloped regions so that limitedoral health resources can be targeted to those who are deemed most inneed of care; thereby supporting cost-effective community-based healthprograms.

2) A visual high throughput (HTP): this more precise test version wouldbe compatible with multi-analyte technologies, but would still enablequantitation of caries risk leading to prediction of future cariesexperience. This test might be administered in a dentist's office where,in combination with other wellness tests, appropriate countermeasurescould be initiated if warranted.

3) A high throughput (HTP) semi-analytical visual test that could beused on archived salivas without expensive quantitation devices

4) A full analytical test with HTP characteristics. This multi-analytetest requires a capture step involving antisera or lectins.

In a preferred embodiment, the test strip has a uniform design thataccommodates, as many ages, races and ethnicities as possible.

In one embodiment, two or three concentrations of a known ligand (i.e.,binding partner) of the caries-predictive lectins are incorporated abovethe matrix to serve as standards or controls. As used herein, the term“ligand” or “binding partner” refers to a member of a pair of moleculesand/or compositions capable of recognizing a specific structural aspectof another molecule or composition, wherein the binding partnersinteract with each other by means of a specific, noncovalent or covalentinteraction.

One example of test version of this invention can be used in anon-clinical setting to provide a general forecast of cumulative cariesexperience, as well as to assess the risk of future caries development(e.g., high, medium, low, very low risk, or zero, for future cariesdevelopment). This version would also be appropriate for use inunderdeveloped regions, so that limited oral health resources can betargeted at those who are deemed most in need of care, therebysupporting cost-effective community-based health programs.

Another test version of the present invention can be used to quantitatedental caries risk leading to the prediction of future caries-experienceat subsequent ages. This test can be administered in a dentist's officewhere appropriate countermeasures could be initiated.

Yet another test version is diagnostic and used with medicallycompromised patients, such as those suffering from diabetes or AIDS.Still another test version features multiple sample, high throughputcharacteristics. The use of this test version would be used to screeningpopulations of saliva samples, such as those used for epidemiologicalsurveys.

The present invention also provides a diagnostic kit for detecting adisease, comprising: a) a means for collecting a saliva sample; b) ameans for measuring the amount of a lectin-binding component in saidsample; and c) an oral fluid standard for comparing the amount of saidcomponent in said sample. In one embodiment of the present invention,the kit is a Western blot format. In kits of this nature, a drop of asaliva sample is transferred onto an immobilizing matrix (e.g.,nitrocellulose filter). The drop is then probed with one or morelectins, and the lectins can then be detected by means of suitablemethods.

III. METHOD FOR PREVENTING DISEASES

The present invention also provides methods for preventing or reducingthe risk of diseases. In particular, the compositions and methods of thepresent invention can be used for preventing oral diseases andassociated diseases. Once symptoms of associated diseases (e.g.,cardiovascular and respiratory diseases) are detected, treatment isdifficult and expensive. Thus, treatment results would be much better ifindividuals could be determined to be at risk prior to symptoms. In thismanner, preventive measures could be taken and early interventionstrategies could be employed.

In one embodiment, the present invention provides a method forpreventing or reducing the risk of a disease in a subject, comprisingproviding an unfractionated saliva sample from a subject; contacting analiquot of the saliva with one or more lectins under conditions thatallow the one or more lectins to bind to a lectin-binding component ofthe saliva; detecting the amount of bound lectin; comparing the amountof bound lectin to the amount known to bind a saliva sample from acontrol subject, wherein the amount is proportional to the risk of adisease in said subject; and administering a therapeutic reagent to thesubject when the content of the component in the saliva is above orbelow the level expressed in normal control (i.e., a subject free fromthe disease being tested for). In some embodiments, the normal controlcomprises an oral fluid standard.

A. Oral Fluid Standards

Various oral fluid standards for testing, calibration andstandardization of devices and methods for the analysis of oral fluidsare well known in the art (See e.g., U.S. Pat. Nos. 5,736,322 and5,695,929 to Goldstein, incorporated herein in their entirety byreference). U.S. Pat. No. 5,736,322 describes oral fluid standardscomposed of an aqueous solution of a mucin and a protease inhibitor. Apreferred oral fluid standard additionally includes an amylase. Anyprotease inhibitor that reduces or eliminates proteolytic activityassociated with a mucin is suitable. Preferred protease inhibitorsinhibit the papain-like (cysteine) proteases. Particularly preferredprotease inhibitors include, but are not limited to, leupeptin,antipain, benzamidine, chymostatin, pepstatin A, and aprotinin. In aparticularly preferred embodiment, the mucin is present at aconcentration ranging from about 0.001% to about 0.4% (w/v); the amylaseis present at a concentration ranging from about 0.1 g/L to about 5.0g/L; and the protease inhibitor is present in a concentration sufficientto reduce or prevent proteolysis of antibodies added to the oral fluidstandard.

The oral fluid standards can additionally include one or more componentsselected from the group consisting of magnesium, calcium, sodium,phosphate, chloride, potassium, and bicarbonate. The oral fluid standardcan additionally include a preservative, most preferably a preservativeselected from the group consisting of thimerosal, gentamycin,chlorhexidine digluconate, and polyhexamethylenediguanide.

The standard oral fluid standard can include serum, more preferablyhuman serum. The serum can be positive or negative for an analyteincluding, but not limited to any of the above-identified analytes. Aparticularly preferred oral fluid standard includes nitrite at aconcentration ranging from about 0.1 mM to about 2 mM; magnesium at aconcentration ranging from about 0.03 mM to about 0.6 mM; calcium at aconcentration ranging from about 0.5 mM to about 5.0 mM; sodium at aconcentration ranging from about 2 mM to about 80 mM; phosphate at aconcentration ranging from about 1.8 mM to about 25 mM; chloride at aconcentration ranging from about 10 mM to about 56 mM; potassium at aconcentration ranging from about 10 mM to about 40 mM; and bicarbonateat a concentration ranging from about 2 mM to about 35 mM. This standardcan additionally include a preservative.

Similarly, the oral fluid standards can additionally include one or moreanalytes. Suitable analytes include, but are not limited to an antibodyselected from the group consisting of an antibody to HIV-1, an antibodyto HIV-2, an antibody to HTLV-1, an antibody to HTLV-2, an antibody toHelicobacter pylori, an antibody to hepatitis A, an antibody tohepatitis B, an antibody to hepatitis C, an antibody to measles, anantibody to mumps, an antibody to rubella, cotinine, cocaine,benzoylecgonine, benzodiazapine, tetrahydrocannabinol, theophylline,phenytoin, β-hCG, thyroxine, thyroid stimulating hormone, folliclestimulating hormone, luteinizing hormone, glucose, insulin, orcholesterol.

U.S. Pat. No. 5,696,929 to Goldstein also describes a saliva standardfor measuring the efficacy of saliva collection kits and for comparingand standardizing analytical methods. Generally, the inventivesubstitute saliva standard has the composition (ingredients presented asmmol/liter): Nitrite 0.1-0.2; Magnesium 0.15-0.6; Calcium 0.5-0.47;Sodium 2-80; Phosphate 1.5-25; Chloride 10-56; Potassium 13-40;Bicarbonate 2-35; Thimerosal 0.01-0.1 g/100 ml; Amylase 0.025-0.1 g/100ml; Mucin (5%) 0.02-0.5 g/liter; Antipain 0.05 mg/liter; Deionized WaterQS to 1 L (approx. eq. 998 ml).

According to one embodiment, in order to test a particular assay, agiven amount of the substitute saliva standard is spiked with apredetermined amount of analyte, the desired dilution made, and then theassay is run. The substitute saliva standard could be spiked with, e.g.,HIV antibody-positive serum, HIV antibody-negative serum, or any othertarget analyte which would ordinarily be detectable in human saliva.Representative of such analytes are those mentioned in theaforementioned U.S. Pat. No. 5,103,836 (incorporated herein in itsentirety by reference).

B. Anti-Caries Reagents

Various anti-caries reagents well-known in the art can used to practicethe methods of the present invention. For example, U.S. Pat. No.6,136,298 to Gaffar, et al. (incorporated herein in its entirety byreference) describes oral compositions containing a substantially waterinsoluble noncationic antimicrobial agent, such as triclosan or xylitolfor inhibiting S. mutans and dental caries. Typical examples of waterinsoluble noncationic antibacterial agents which are particularlydesirable from considerations of effectiveness, safety and formulationare: halogenated diphenyl ethers; benzoic esters; sesquiterpene alcoholssuch as farnesol, nerolidol, bisabolol, santalol and like compounds;halogenated carbanilides; and phenolic compounds (including phenol andits homologs; mono-, poly-alkyl and aromatic halo-phenols; resorcinoland catechol and their derivatives; and bisphenolic compounds. Thenoncationic antibacterial agent is present in the dentifrice in aneffective antiplaque amount, typically about 0.01-5% by weight,preferably about 0.03-1.0% by weight and most preferably about .0.3-0.5%by weight. The antibacterial agent is substantially water-insoluble,meaning that its solubility is less than about 1% by weight in water at25° C., and can be even less than about 0.1% by weight.

The preferred halogenated diphenyl ether and most preferred noncationicantibacterial agent is triclosan. Preferred other noncationicantibacterial agents are hexyl resorcinol and 2,2′-methylene bis(4-chloro-6-bromophenol). Xylitol, when present in amounts ranging fromabout 0.1% by weight to about 40% by weight, also enhances theantibacterial and anticaries properties of the oral compositionsdescribed above.

U.S. Pat. No. 5,807,541 to Aberg, et al. (incorporated herein in itsentirety by reference) describes compositions and methods for inhibitingthe development of caries using non-steroidal anti-inflammatory drugs(NSAIDs) and fluoride reagents. NSAIDS can be characterized into fivegroups: (1) the propionic acids; (2) the acetic acids; (3) the fenamicacids; (4) the biphenylcarboxylic acids; and (5) the oxicams.

Propionic acid NSADs are non-narcotic analgesics/nonsteroidalantiinflammatory drugs having a free —CH(CH₃)COOH group, whichoptionally can be in the form of a pharmaceutically acceptable saltgroup, e.g., —CH(CH₃)COO⁻Na⁺. The propionic acid side chain is typicallyattached directly or via a carbonyl function to a ring system,preferably to an aromatic ring system. Exemplary propionic acid NSAIDSinclude: ibuprofen, indoprofen, ketoprofen, naproxen, benoxaprofen,flurbiprofen, fenoprofen, fenbufen, pirprofen, carpofen, oxaprozin,pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofen,fluprofen, and bucloxic acid. Structurally related propionic acidderivatives having similar analgesic and antiinflammatory properties arealso intended to be included in this group.

Acetic acid NSAIDs are non-narcotic analgesics/nonsteroidalantiinflammatory drugs having a free —CH₂COOH group (which optionallycan be in the form of a pharmaceutically acceptable salt group, e.g.,—CH₂COO⁻Na⁺) typically attached directly to a ring system, preferably toan aromatic or heteroaromatic ring system. Exemplary acetic acid NSAIDSinclude, but are not limited to, ketorolac, indomethacin, sulindac,tolmetin, zomepirac, diclofenac, fenclofenac, alclofenac, ibufenac,isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac,clidanac, oxpinac, and fenclozic acid. Structurally related acetic acidderivatives having similar analgesic and antiinflammatory properties arealso intended to be encompassed by this group.

Fenamic acid NSAIDs are non-narcotic analgesics/nonsteroidalantiinflammatory drugs having a substituted N-phenylanthranilic acidstructure. Exemplary fenamic acid derivatives include mefenamic acid,meclofenamic acid, flufenamic acid, niflumic acid, and tolfenamic acid.Biphenylcarboxylic acid NSAIDS are non-narcotic analgesics/nonsteroidalantiinflammatory drugs incorporating the basic structure of abiphenylcarboxylic acid. Exemplary biphenylcarboxylic acid NSAIDsinclude diflunisal and flufenisal. Oxicam NSAIDs are N-aryl derivativesof 4-hydroxyl-1,2-benzothiazine 1,1-dioxide-3-carboxamide. Exemplaryoxicam NSAIDs are piroxicam, sudoxicam and isoxicam.

Certain histidine-rich polypeptides (“HRPs,” also referred to ashistatins) having a substantial proportion L-histidine (i.e., betweenabout 14 and 40 mole and amino acid residues), have antibacterial andantifungal properties, particularly against S. mutans and Candidaalbicans. (U.S. Pat. No. 4,725,576 to Pollock, et al.). HRPs areadministrable to the loci of infection, particularly in the oralsurfaces. Delivery can be by any conventional means, preferably topicalmeans. In the case of oral administration, this would includedentifrices; mouthwashes; denture washes or soaks; denture adhesives orcements; and incorporation into polymers associated within the denture,particularly with the interface of the denture with the gum.Histatin-based peptides having antibacterial and antifungal propertiesare also described in U.S. Pat. Nos. 5,912,230; 5,885,965; 5,631,228;5,646,119; and 5,486,503 to Oppenheim et al., each of which isincorporated herein in its entirety.

U.S. Pat. No. 5,801,226 to Cummins, et al. (incorporated herein in itsentirety by reference) describes sodium and stannous fluorides,aminefluorides, monosodiumfluoro-phosphate, casein, and plaque bufferssuch as urea, calcium lactate, calcium glycerophosphate, strontiumpolyacrylates, as anti-caries reagents.

U.S. Pat. No. 5,013,542 to Hay, et al. (incorporated herein in itsentirety by reference) describes compositions containing non-immunogenicamino acid segments of proline-rich proteins for inhibiting the adhesionof disease-causing microorganisms to tooth surfaces. Such microorganismsinclude, but are not limited to S. mutan, S. sanguis, S. sobrinus,Actinomyces viscosus, and Bacteroides gingivalis. The amino acid segmentcan be obtained from acidic, proline-rich proteins, such as thosederived from human saliva. These proline-rich proteins show markedcharge, structural asymmetry and exceptional reactivity to apatiticsurfaces. When intact, these proline-rich proteins also promote theadhesion of microorganisms to apatitic surfaces. Because they arederived from human proline-rich proteins, they are recognized as “self”by humans, and antibodies to them have not been reported in humans. Themineral-binding segments can be used as the active ingredients alone orin combination with the other compounds, such as enzymes, antimicrobialagents, etc., in various compositions used for the treatment of theteeth so as to limit the adhesion and/or growth of microorganisms.

The active ingredient can be derived from segmenting a natural orsynthetic, proline-rich protein, to provide a non-immunogenicingredient. The non-immunogenic amino acid segment can be obtained byvarious techniques, such as by cloning, or by synthesizing analogs ofthe natural molecules or their segments by chemical means. Thenon-immunogenic amino acid segment can also be obtained enzymatically orby cleaving the proline-rich protein derived from human saliva by theenzyme trypsin. The removed portion of the proline-rich protein containsthe bacterial binding sites. A variety of human, proline-richphospho-proteins can be employed.

U.S. Pat. No. 6,231,857 to Shi, et al., incorporated herein in itsentirety by reference, describes antibodies of S. mutans, which can beused in treating dental caries. Specifically, Shi, et al. describe threemonoclonal IgG antibodies, each of which specifically binds an antigenon the surface of S. mutans. One monoclonal antibody is produced by ahybridoma deposited with the American Type Culture Collection as ATCCNo. HB12559, and is designated SWLA1. A second monoclonal antibody isproduced by a hybridoma deposited with the American Type CultureCollection as ATCC No. HB 12560, and is designated SWLA2. The thirdmonoclonal antibody is produced by a hybridoma deposited with theAmerican Type Culture Collection as ATCC No. HB 12258, and is designatedSWLA3.

IV. RESULTS

The methods of this invention for predicting the risk of a disease in asubject provide a method for assessing the risk of the disease accordingto risk levels such as high, medium, low, very low, or zero. Forexample, in certain populations, 0-2 DFS may be associated with a verylow risk of developing caries in young adults, while 3-8 DFS may beassociated with low risk, 9-16 DFS with medium risk, and 17 or more DFSwith high risk. Thus, for example, according to one embodiment, if theamount of bound lectin is comparable to that for the “very low riskgroup,” it can be predicted that this individual is at risk ofdeveloping 0-2 DFS.

In another embodiment, the methods of this invention for predicting therisk of a disease in a subject can further comprises assessing the riskof future development of the disease in the subject. For example, themethod of assessing future risk can comprise comparing the amount oflectin binding to a regression analysis derived from a group of subjectsexpressing a range of disease severity.

In one embodiment, the methods of the present invention provide acorrelation between the amount of lectin that binds to a saliva sample,combined with age and gender information, with the number of dentaldecays (e.g., early-onset dental caries, adult dental caries, rootcaries, DFT, DMF, DMFS, dfs, dft, dmft, dmfs, and dfs/t). The R² of theregression analysis is preferably at least 0.3 (i.e., any number betweenand including 0.3 and 1). For example, for a 3-level test, the R² may beat least 0.9; for a 4-level test, the R² may be at least 0.98; for a5-level test, the R² may be at least 0.99.

In general, according to one method of this invention, a single dropletof saliva is applied to a matrix membrane. This and the standard dots(i.e., saliva samples from a control subject) are then probed with asingle lectin or a specific panel of lectins that work together as amixture to give a single visual test report. The standards arecalibrated to be appropriate for as many unique groups as are found. Inone embodiment, the test is universalized to accommodate all groupsusing a test of uniform design.

The amount of bound lectin is visualized, for example, with coupledenzymes, such as alkaline phosphatase or horseradish peroxidase, by acoupled fluorochrome, such as FITC or AMCA, or through the use oflectins that are coupled to colored microparticles. In one example, thelectin result, combined with age and gender, provided a correlation withDFT that has an R² of 0.90 for a group of individuals that include 7-9year-old Chinese girls and Hispanic boys as well as 20-25 year-oldAsians and Caucasians of both sexes.

The lectin test with the 7 to 25 age span, though forecasting thecorrect number of DFT, may not be useful for predicting individual risklevels because it simply predicts the number of DFT, regardless of age.For instance, this particular regression equation placed the 25 year-oldwith five DFT above the 7-9 year-olds with four DFT in spite of thelikelihood that the former was in the low to medium DFT for his agegroup and the latter in the high group for their age. However, if age isremoved from the regression equation and the Hispanic and Chinese 7-9year-olds are considered together as a group and the 20-25 year-oldChinese and Caucasians considered as a group, the following was achievedwith the panel of three lectins and gender. The regression equation forthe children gave an R² of 0.972 and a P-value of 2.96×10⁻⁵ against afour-level risk assessment system equivalent to high, medium, low, andvery low. The regression equation for the young adults gave an R² of0.983 and a P-value of 6.18×10⁻⁴ against the same four-level riskassessment system.

Several different test formats can be used for specialized applications.For instance, one test version could be used in a classroom setting toprovide a general expectation of caries experience (such as high,medium, low, or very low) for the dentally indigent student, as well asto assess the risk for future caries development, This version wouldalso be appropriate for use in underdeveloped regions so that limitedoral health resources can be targeted to those who are deemed most inneed of care; thereby supporting cost-effective community-based healthprograms. Another more precise test version would be compatible withmulti-analyte technologies, but would still enable quantitation ofcaries risk leading to prediction of future caries experience. This testmight be administered in a dentists office where, in combination withother wellness tests, appropriate countermeasures could be initiated ifwarranted. A third version would be a high throughput (HTP)semi-analytical visual test that could be used on archived salivaswithout expensive quantitation devices. A fourth version would be fullanalytical with HTP characteristics. The use of this variant of theoriginal test would be targeted to screening populations of salivasamples, such as might be envisioned for epidemiological surveys as wellas for discovery.

In addition, the use of this test could apply to the risk assessment ofadditional diseases with which caries experience is correlated or cariesexperience is found to be an indicator of, or associated with, theirrisk.

In pre-teenagers, teenagers, and young adults, there are not enoughteeth and/or enough exposure time for the teeth to achieve high enoughcorrelations to support individual risk prediction, simply by countingthe number of cavities. The method of the present invention bridgesthese ages of uncertainty by providing significant individual diagnosesof caries risk that could lead to earlier intervention and prevention.

V. Advantages of the Present Invention

The methods, test devices, and diagnostic kits of the present inventionpresent the following advantages over convention methods and testdevices. First, scientific evidence suggests that the MUC7 mucinconcentration is likely to be one of the key determinants of the S.mutans titers in saliva. Thus, the present invention allows theprediction and diagnosis of the cariogenesis process at an earlier stagethan S. mutans titer alone, and provides more avenues of prevention.

Second, the experimental results of the present invention show a clearnumerical relationship to caries experience, in contrast to currentlyavailable technology for detecting S. mutans such as DENTOCULT® StripMutans (“SM”) test strips (manufactured by Orion Diagnostica, Finland).At best, the DENTOCULT® SM test strips can differentiate the S. mutanstiters in saliva into categories of high, medium, low and none. Thepresent invention is also advantageous over DENTOCULT® strips because ofthe simplicity and ease of use. In a preferred embodiment, the methodsof the present invention can be evaluated in a nonclinical setting bynon-technical personnel. In contrast, the DENTOCULT® SM strips must becultured under sterile conditions and evaluated by a trained,experienced personnel.

The present invention also provides non-invasive compositions andmethods for predicting and diagnosing the risk of a disease in asubject. Numerous analytical methods have been developed for determiningthe presence or absence of, and/or quantifying the amount of variousanalytes in tissues and fluids of organisms. Currently, most diagnostictesting is done with either blood, urine, fecal material, or tissuebiopsy. Testing based on these materials, however, entails substantialinvasion of privacy, and poses a significant safety hazard (particularlywith testing of blood). In contrast, the collection of oral fluid fortesting, including saliva and/or mucosal transudate, entails relativelylittle invasion of privacy, is relatively safe, and can be accomplishedrapidly with relative ease.

Furthermore the methods and test devices of the present inventionprovide new diagnostic tests for early disease detection, definingindividual patient risk of adverse response to drugs, monitoringtherapeutic progress, and determining outcomes of treatment. The salivadiagnostic methods and kits of this invention have provide selectivity,sensitivity, appropriate response time, dynamic range (values ofinterest), representative sampling, reliability or stability as well asthe ability to assess multiple substances simultaneously.

The mucin test, as described in U.S. Patent Publication No.20003/0040009 A1, does not apply to all races or ethnicities. Thepresent invention addresses this issue by providing a universal testwhich can forecast equally well the accumulated caries history, i.e.,DFT (decayed and filled permanent teeth) among various races and agegroups. For example, in one embodiment of the invention, the testforcasted equally well the accumulated caries history in Hispanic andChinese 7-9 year-old children and in Asian and Caucasian 20-25 year-oldadults.

The dental examiner cannot reliably identify those who are high or lowrisk for future caries development simply by counting the number ofcaries. This is especially true throughout the late pre-teen, teenage,and early adult years, simply because the range of DFT is too narrow toachieve statistical significance for distinguishing the different levelsof caries experience. The present invention provides this missing pieceof diagnostic information. This leads to earlier, better, moreindividualized treatment planning. The outcome of the test will alsoprovide a rationale for individualizing the frequency and aggressivenessof preventive measures. With the aid of this tests described herein, thedental examiner will be able to assume the role of oral health caremanager to the extent that their individualized intervention could leadto abolition of new caries development even in those who are most atrisk.

Another application of this invention is in so-called third worldenvironments, where the tests of the present invention can be used toidentify the highest risk individuals in non-clinical settings bynon-clinicians. This identification of high-risk children and youngadults could lead to targeted treatment, maximizing the effectiveness oflimited resources.

The following examples serve to explain and illustrate the presentinvention. The examples are not to be construed as limiting of theinvention in anyway. Various modifications are possible within the scopeof the invention.

EXAMPLE 1

The first objective of this study was to confirm and extend theinventors' original findings from the 1991 young adult dataset to acontemporary group of similar composition, recruiting 80-100 students atthe USC School of Dentistry.

The second object of this study was to perform a similar study in 7-8year-old children to learn if the correlations between DFT and mucinconcentrations seen in young adults also apply. Analysis of this dataprovided new insights that are reflected in Example 2.

The third objective was first to better understand the component of MUC7and MUC5B mucins which appear to have the best association with theforecast of DFT. A second aspect of this aim was then to incorporatethese elements into a prototype test with commercial potential.

A. Carbohydrate Studies Leading to Identification of the Most PredictiveElement(s) Related to MUC7 and MUC5B Mucin Concentrations.

1. Dot blots: The first objective was to evaluate whether the broaderattributes, such as total sialic acid, total carbohydrate, and totalapomucin, of the mucins in saliva against a panel of individuals whoseDFT and mucin content, as measured by Stains-all, are consistent withthe correlation that was noted in young adult Caucasians. A secondobjective was to use a library of lectins to determine a connectionbetween the content of Lewis and T antigens known to be present on MUC7mucin in varying amounts, and the caries-prone and caries-resistantindividuals identified by our test. This library of lectins alsoincluded the potential to distinguish α-2,6 and α-2,3 linked sialicacids.

A pilot experiment, using 0.2 μL of whole saliva, quickly revealed thatthe lectin study is a promising, simple approach to quantitating themain factor associated with caries-resistance. The results of thisexperiment are shown in FIG. 1. The lectin panel (Vector Laboratories)included Jacalin (Jackfruit seed lectin) and ACL (Amaranthus CaudatusLectin) for different configurations of sialylated T-antigen, AAL(Aleuria Aurantia Lectin) and UEA I (Ulex Europaeus Agglutinin I) fordifferent Lewis antigens, SNA (Sanbucus Nigra Lectin) primarily forα-2,6 linked sialic acid with some α-2,3 activity, and MAL I (MaackiaAmurensis Lectin I) for α-2,3 linked sialic acid. The saliva of the twoboys also contained substantially different levels of mucins (MUC7 at3078 U/mL [subject #15] vs. 407 U/mL [subject #27], and MUC5B at 3725U/mL [subject #15] vs. 696 U/mL [subject #27]). This pilot studysuggested that the quantity of α-2,3 linked sialic acid in whole salivamight embody a DFT-forecasting potential similar to that of Stains-allbinding to the two mucins. Indeed, the intensity difference between thespots for subjects #15 and #27. which is 6.9x, and the differencebetween their combined Stains-all derived mucin concentrations, which is6.2x, are very similar. Differences in the other lectins can also beseen.

The panel of lectins was repeated with the same two salivas at theequivalent of 0.2, 0.1, 0.05, and 0.02 μL diluted to equivalent spottingvolumes of 5.0 μl. This provided a concentration series for each lectinand subject. Linear regressions obtained for all of the lectins with thesubject #15 saliva had an R² of >0.93. The relative differences that arereported have been repeated within the limitations of the dot blotsystem. The results of this experiment are summarized in Table 1. Inthis and the experiments that follow, the 0.2 μL intensity value fromthe regression of each lectin with Griffin #15 saliva is assigned avalue of 100 for convenience and all other samples are normalizedrelative to that value and volume of saliva. TABLE 1 Evaluation oflectin panel with Griffin subjects #15 and #27 Griffin #15 Griffin # 27JAC 100 320 ACL 100 100 AAL 100 56.5 UEA I 100 26.5 SNA 100 29.4 MAL I100 14.4

This experiment confirms the contention from the previous experiment,that the amount of α-2,3 linked sialic acid is the most distinctivedifference between the two salivas. The SNA result further supports theprominence of sialic acid. AAL and UEA I suggest that the amount ofLewis antigen, especially the variety with α-1,2 linked fucose, maysomehow be interwoven in the relationship between mucin and DFT. On theother hand, the quantity of the form of T-antigen that is recognized byJacalin may be a negative factor. Based in part on this experiment, ACLwas dropped from the lectin panel and MAA (Maackia amenurensis, EYLaboratories) was added for evaluation of the broader panel of salivas.MAA, like MAL I, also shows a preference for α-2,3 over α-2,6 linkedsialic acid, but it has other differences that may be useful in ourapplication.

A panel of salivas was assembled from five different groups and spansthe time period from our first study (1990) to the present. Half of thepanel members exhibited an inverse relationship of MUC7 mucinconcentration to DFT, and the remainder exhibited a direct relationship,e.g., high mucin and high caries or low mucin and low caries. Thecharacteristics of each subject and their relative responses to the sixlectins are shown in Table 2.

Saliva standards indicate that the mucin concentrations have remainedstable since the 1990 study as long as they were kept frozen. The lectinassay system was simple and lacked the precision of an establishedanalytical system, but provided repeatable data on a relative scale.

Griffin subject #15 provided a relative standard curve for every lectinusing the following amounts of saliva: 0.2, 0.1, 0.05, and 0.02 μL. Allspotting volumes were 5.0 μL. The sample was dried on nitrocellulose andreacted with the lectin-biotin complex. After washing, thenitrocellulose was bathed in avidin-alkaline phosphatase. Colordevelopment was with NBT/BCIP that was allowed to continue until therewas a range of color intensities. Quantitation was accomplished bymeasuring the average color intensity on a transect of the diameter ofthe dot blot. TABLE 2 AGE/ RACE/ MUC7 MUC5B DFT S. MUTAN JAC AAL UEA ISNA MAL I MAA YEAR STUDY SUB. # SEX ETHNIC resting resting (H, L) (H. L)T-antig. Lewis Lewis α-2,6 α-2,3 α-2,3 1991 Aging 8  26/M Cauc 1505 344L — 107 62.9 40.2 51.6 44.9 78 17  21/F Cauc.  170 922 H — 56 2.3 0 20.00 10 2000 S. mutans 3 >65/? Cauc.    <6 281 — H 270 8.2 19.3 18.3 37.522 7 >65/M Cauc.  805 624 — L 240 88.7 12.8 39.6 167.5 42 2002 Griffin 3  7/F Chin. 1863 1082  H — 200 42.1 12.3 47.6 19.5 70 4   8/F Chin. 1191289 H — 559 53 21.8 49.7 6.3 58 5   7/F Chin. 1438 160 H — 421 39.6 14.929.9 14.3 10 10   7/F Chin.  631 504 zero — 1034 55 21.8 52.6 23.8 45 13  7/F Chin.   7 612 zero — 399 20.5 21.9 52 30.5 36.5 15   7/M Hisp.3078 3725  zero — 100 100 100 100 100 100 27   8/M Hisp.  407 696 H —320 56.5 26.5 29.4 14.4 2002 USC 4 21/F Asian 2192 2743  H — 354 76.62.2 61.3 8/5 70 35 26/M Pac. Isl. 765?  20 H — 78.1 6.8 2.4 10.7 7.0 9.542 23/F Asian 57677  2048?  zero — 221 63.7 12.7 51.6 70.5 82

The patterns of reactivity are different for each lectin. The high orlow DFF assignment was based on the relative numbers of DFT within eachsubject's peer group. The high titer of S. mutans referred to >10⁶ andlow to <10⁵ cfu. The salivas from the S. mutans study were not includedin the following calculations because of subject age and lack of accessto their DFT. The degree of correlation with DFT (H or L) differs foreach lectin and is shown for the remaining salivas in Table 3. TABLE 3Regressions and Correlations Analyzing the Lectin Affinities to WholeSaliva Reported in Table 2 Variable(s) R R² Adj. R² P-value A. MUC7 +MUC5B vs. DFT −0.35 0.12 0.00 NS (H/L) B. MAL I vs. DFT (H/L) −0.76 0.570.53 0.004 C. SNA vs. DFT (H/L) −0.59 0.34 0.28 0.05  D. MAL I + SNA vs.DFT (H/L) −0.76 0.57 0.48 0.02  E. MAA vs. DFT (H/L) 0.16 NS F. UEA 1vs. DFT (H/L) 0.22 <0.10-->0.05 G. AAL vs. DFT (H/L) 0.06 NS H. JAC vs.DFT 0.00 NS I. MAL I + JAC vs. DFT (H/L) −0.83 0.69 0.62 0.005 J. MALI + JAC + AAL vs. DFT −0.89 0.78 0.70 0.005 (H/L)

The diverse nature of the saliva panel is evidenced by the fact thatneither the MUC7 or MUC5B mucin nor the combination of the two has but aweak, non-significant correlation with DFT (Table 3, row A). MAL I showsthe highest individual correlation with DFT and except for a relativelynarrow range of lectin reactivity between high and low DFT, there is nooverlap (Table 3, row B). SNA shows the next best individual correlationwith DFT (Table 3, row C), but in combination with MAL I, the strengthof the relationship to DFT is not improved (Table 3, row D). MAA, UEA I,and AAL all exhibit measurable correlation, but do not achievesignificance (Table 3, rows E-G). Jacalin (JAC) is not correlated withDFT (Table 3, row H), however it does appear to contribute substantiallyto the correlation when included with MAL I (Table 3, row I). Inclusionof AAL in the multiple regression yielded the highest R² observed withthe lectins alone (Table 3, row J).

Interestingly, MAL I, SNA, MAA, UEA L and AAL are all significantlycorrelated with each other. JAC is not significantly correlated with anyof the other lectins. These results suggest that the sialicacid-specific lectins are primarily associated with Lewis antigens andnot T-antigen. However, none of the other lectins improve correlationwhen combined with MAL I. The exception is that after MAL I and JAC arecombined then AAL contributes a further substantial improvement (Table3, rows I and J).

When compared with the mucin concentrations, SNA, MAA, AAL, and MAL Iare each significantly correlated with MUC5B mucin concentration. OnlyMAA, AAL, and MAL I showed correlation with MUC7 mucin concentration. Inthe unusual combination of MAL I reactivity and MUC5B concentration,there was an improvement of the overall relationship to DFT to an R² of0.59.

The potential for one of the mucins to improve the regression equationled us to broaden the search to include age and gender, as well as thetwo mucins. The result was that a combination of independent variableswas found that yielded an R² of 0.932. The significance of theregression equation was p=0.015 with satisfaction of normality, constantvariance, and power tests. The independent variables followed by theirrelative contribution to the regression equation in parentheses are MAL(49%), JAC (6%), MAA (3%), MUC7 mucin (14%), MUC5B mucin (6%), gender(10%), and age (12%). By standardizing the contribution of eachindependent variable with the regression formulas, and then taking thesum of these values for each individual, the complex outcome of therelationship can be visualized by a simple linear regression graph asshown in FIG. 2. The graph illustrates that there are no overlapsbetween high and low caries experience for children and young adults ofboth genders and at. least three different ethnicities, Chinese,Hispanic, and Caucasian. The population confidence interval wasprogrammed at 98%. Thus, given the above information, a prediction ofhigh or low caries experience can be made with high accuracy. FIG. 2also demonstrates the potential for insertion of a highly significantmiddle range of caries experience as more data becomes available. Thedata shown in FIG. 3 demonstrates that the information needed to predictcaries experience with high probability is still contained in the dataeven though the individuals represented near opposites with regard toMUC7 mucin concentration.

Development of an analytical test for the high throughput applicationthat uses multiple independent data inputs requires the testing ofmultiple analytes on an automated format. The MAL I affinity for asubset of α-2,3 linked sialic acids provides a strong link to theforecast of DFT. An alternative approach to the strip test is the dotblot approach, as used above, which works with whole, unfractionatedsaliva. This greatly simplifies development, manufacture, packaging, andstorage procedures. A preferred test strip provides R²'s of 0.90 orbetter.

The information obtained from this Example suggests the possibility thatwhereas the two mucins are the primary carriers of the MAL I reactivityin Caucasians, in Asians there may be different molecules involved.Western blots analysis described in Example 2 provided definitiveinformation in this regard.

B. Tests for simple, reliable disclosants for the mucin's predictiveelement(s) that can be adapted to the above tests. The inventors'experience with quantitating dot blots demonstrated that this representsa greater potential for developing future tests than the PAGE approachthat has been used extensively to this time. FIGS. 4 and 5 illustratethe linear potential, as well as a metachromatic color option, in amodel system that used mouse sublingual mucin. As far as the MUC7 mucinrelationship to DFT in Caucasian young adults, only a three-folddifference in concentration was needed to distinguish the threesignificant classes of caries experience. The dot blot can easily becalibrated to distinguish the two threshold concentrations required toassess the three levels of risk.

FIG. 6 shows an example of the dot blot format. Using salivas from thetwo Hispanic boys featured in FIG. 1 and Table 1, a dried droplet oftheir whole saliva was probed with MAL I-biotin followed by avidin-AMCA.The result is a direct digital image of the fluorescence produced bylong wave UV irradiation. The difference is striking and representsapproximately a six-fold difference. The four-fold serially dilutedhomologous saliva samples also reveal easy differences to distinguishvisually.

EXAMPLE 2

The purpose of this study was to “universalize” and complete developmentof several iterations of a simple, non-invasive saliva test not only forforecasting individual accumulated caries experience but, moreimportantly, for assessing levels of risk for future caries development.The test can be integrated into dental practice as a common diagnosticprocedure health screening, and in broader oral health campaigns toimprove identification, treatment, and prevention in high-riskindividuals. The diagnostic screening information provided by thesecombinations of test and technology will aid the health care provider inidentifying caries-prone children, teenagers, and young adults. Thiswill be most helpful at stages of dental development when physicalexamination alone or dmfs (decayed, missing, and filled surfaces) indeciduous teeth and DMFS in permanent teeth cannot identify withstatistical certainty, those individuals who are at-risk.

Additional factors in saliva, which may or may not be associated withmucins, were identified and evaluated by lectin affinities for theproperty of caries prediction, and then were developed into a cariesrisk test suitable for commercialized. The assays provided factors thatare better and more broadly applicable risk indicators than MUC7 andMNC5B mucins alone. With these factors, groups of children and youngadults, for whom the mucins may not be forecasters of accumulated cariesexperience, can still be identified with high probability, even whengrouped together with individuals for whom the mucins are indicators.These additional factors can be quantitated with less difficulty thanthe mucins and present greater opportunity for developing simple,reliable tests.

A. Background

1. Epidemiological studies leading to caries risk prediction: There havebeen numerous models generated for caries risk assessment, and they arerarely identical in design. A review of the caries risk assessmentliterature through 1989 concluded that: 1) clinical variables werebetter predictors, 2) the most significant indicator was past cariesexperience, and 3) regression analysis was the preferred statisticalapproach (Newbrun, E. and Leverett, D., Risk Assessment in Dentistry,Bader, J. D., ed. Chapel Hill: University of North Carolina DentalEcology, 1990, p. 304; Powell, L. V. Community Dent. Oral Epidemiol.26:361-371 (1998)). The literature published between 1989 and 1997confirmed the preeminent role of past caries experience in caries riskprediction (Powell, supra). The most accurate models for risk assessmenthave also included bacterial levels. Assessing this information from alongitudinal perspective, as reviewed by Powell (supra), the presentinventors noted that past caries experience is the only predictor ofsignificance identified in every age group. Thus, to the extent thatpast caries experience or DFF can predict the level of caries risk, theforecast of past caries experience or DFT is also predictive.

The review by Powell also notes that there have been no caries riskstudies of young adults (18 to 33 years of age). Another relevantobservation was made in the NIH-sponsored epidemiological study entitled“Oral Health of United States Adults” (A. J. Miller, et al., Oral Healthof U S. Adults, NIDR. 87-2868 (1987)). Here it was shown that thedecayed and filled surface measurement (DFS) on coronal surfaces is anaccurate, linear record of initial incidents of caries formation untilabout age 40. Beyond that age, this clinical indicator becomes less andless reliable as an indicator of the long-term rate of caries formation.The subjects included in a study conducted by the present inventors,which harbored the discovery of the strong correlation between salivamucin concentrations and DFT, ranged from 18 to 33 years of age, fillingthe gap in age groups that exists in the literature for prediction ofcaries risk. In addition, it appears that this age group embodies theperiod when the accurate record of first-time caries experienceapproaches its maximum range of expression.

2. The science of caries risk prediction and the multifactorial natureof cariogenesis: A recent review in this area upholds the long-held viewthat cariogenesis is multifactorial (M. Lenander-Lumikari and V.Loimaranta, Adv. Dent. Res. 14:40-47 (2000)). However, equally importantis the principle that correlation does not determine or require anyapparent known causal relationship. The variables tested in many of thecaries risk studies are often far removed from any apparent or knowndirect relationship to cariogenesis (Powell, supra). Thus, because ofthe practical, predictive theme of the proposed project, developing ahypothesis that rationalizes the multifactorial nature of cariogenesisand the high coefficient of determination (R²) between DFT andconcentrations of saliva components is not critical to success. A modelthat explains approximately 9.0% of the variation inl DFT, such ascontained in our report for 24-33 year-olds, does not preclude existingdata suggesting that cariogenesis is multifactorial. The review byLenander-Lumikari and Loimaranta (supra) concludes that other than theearly clinical signs that support caries formation, such as plaquebuild-up and enamel erosion, there is no single factor that can beviewed as having a broad, unified impact on the initiation of cariesformation in individuals. Thus far, only low impact factors have beenidentified, leaving a huge void in quantitative assignment of therelative effects of various factors on cariogenesis. While not wishingto be bound by any particular theory, the inventors believe that mucinsand other components in saliva are important factors that fill thisvoid, permitting regression analysis with significant predictivepotential.

3. Potential for the saliva test for caries risk to improve oral healthcare: The ability to predict the level of risk of future cariesdevelopment creates the opportunity to “fine-tune” prevention.Scientifically based, individually appropriate modalities and spacing ofpreventive treatments, e.g., quarterly vs. semi-annual or annual visits,could be integrated into each patient's long-term treatment/preventionplan. This would be especially appropriate for children and young adultswho otherwise might expect to develop substantial numbers of new cariesas they grow older.

A second area oral of health care that the test could benefit is in the.targeted treatment and prevention of high-risk children and young adultsin third-world environments. Here, targeted application can maximizeeffectiveness of limited resources.

B. Model Three-Level Risk Test Based on Mucin Concentrations.

A model for forecasting accumulated caries experience was developed fromour earlier young adult group. The model was targeted to MUC7 mucinconcentration in the 24-33 year-old group and thus limited in its scope.However, in the course of working with the data to achieve this goal,several strategies became apparent that may be generally applicable tobuilding future models for prediction. The first of these is that thegoal of three non-overlapping zones of significant prediction may beachieved several ways in the same group of individuals. In addition,different combinations of independent variables may be used to achievethe three-level goal in different groups of individuals. In thisparticular regression analysis, there was only a three-fold differencein mucin concentration between high- and low-caries groups. Thus, oncethe analytical data is processed and these thresholds have beenidentified, future tests need to be calibrated only at the two thresholdconcentrations to complete a three-level test. Finally, since mucinconcentrations and DFT are continua in the population, there will belegitimate borderline individuals who must be accounted for by themodel.

The model developed for the 24-33 year-olds is as follows: 1) <400 MUC7mucin units/mL=high risk/caries prone (25%); 2) 400-1200 MUC7 mucinunits/mL=medium risk (58%); and 3) >1200 MUC7 mucin units/nL=low risk(17%). In this example, the threshold concentrations between high andmedium and medium and low DFT coincide with the points at which theparallel confidence interval lines cross over the regression line.Individuals with borderline concentrations of mucin are best placed inthe next higher risk category in order to avoid the error of predictingtoo low, which would have serious health consequence. The percentageslisted above, next to the risk level, are that proportion of the modelgroup who resides within the risk level. The distribution is similar tothat found in the general population (U.S. Dept. of Health & HumanServices. National Institutes of Health Consensus Development ConferenceStatement. Diagnosis and Management of Dental Caries Throughout Life(2001)). The major elements covered in this model can be replicated inany other predictive relationship that is exploited for the caries risktest.

Another phenomenon that was noticed in the young adult group of 12 yearsago was that in those individuals with no detectable MUC7 mucin, DFT wasperfectly correlated (R²=1.00) with age over the range of 21 to 33years. This allowed for calculating a rate of caries development in thisgroup at 0.83 new DFT per year. If this was not a completelyserendipitous occurrence, then we might find in our current subjects, asubgroup that will develop new caries at a relatively rapid, predictablerate. A group such as this would greatly facilitate clinical studies ofpotential caries preventives both in time, number of participants, andlevel of potential statistical certainty.

For any test to proceed to commercialization, the test and theprediction model must be validated. This is accomplished not only byaccepted protocols for “blinding” the second-half phase of datacollection and analysis, but also by analyses that reveal the type,frequency, and magnitude of errors, as well as for the usual measures ofsensitivity and specificity.

C. Test Composition That Achieves a Lectin-Based Three-Level Caries RiskTest

Development of a caries risk test using a single droplet of saliva isextremely attractive because, in addition to simplifying the design anduse of a strip test, it “globalizes” the predictive element(s) in salivaand accommodates the possibility that mucins may not be the onlycarriers of the predictive element. Cases in point are the Griffinsubjects # 3, 4, 5, 10, and 13. These children go completely against theinverse relationship between mucin concentration and DFT identified inthe original study of young adult Caucasians. Subjects 3, 4, and 5 haverelatively high concentrations of mucin and high DFr for their age, andsubjects 10 and 13 have relatively low concentrations of mucins and zerocaries. However, when the saliva panel is screened with lectins, acombination of factors can be assembled that still correctly forecastseach individual's caries load. From this the inventors concluded-thatfactors other than the mucins may also carry the predictor elements andthat there may be individual differences in the molecules that areassociated with these elements. This possibility was explored by Westernblot analyses of the panel salivas, as shown in Table 3 and FIG. 2,using the lectins that have been shown to be contributors to thepredictive regression equations.

The regression equation that is illustrated in FIG. 2 used input fromthree lectins, both mucins, and two “fixed” variables, age and gender.With respect to the analytical test (vs. the strip test), if a dot blothigh throughput format is used, the required lectin information could beacquired by multiple assays as above. The mucin concentrations could beacquired by antibody binding as illustrated in FIGS. 4 and 5. All ofthese assays could use the same avidin conjugated reporter, and all havebeen demonstrated to work within the dot blot format. The fixedvariables are easy to interpret and factor into the analysis leading tothe individual prediction.

The design of the strip test preferably provides simplicity and ease ofuse, while still achieving the three zones of significance. Preferably,the strip test is accomplished with a single spot of whole saliva thatcan be compared to two standard spots, which represent the twothresholds between high and medium and medium and low caries risk.One-step reporting and visual reading are also desirable and arediscussed below. As noted for the analyses with the young adult data,the goal is equaully achievable by different strategies or combinationsof independent variables. In the case of the saliva panel the R² can besubstantially improved to 0.983 from 0.932 (FIG. 2) by inclusion ofethnicity. This provides the opportunity to drop MUC5B concentrationfrom the equation with a relatively small loss of R² to 0.961. An R² of0.90 or higher is the benchmark for achieving the significant threelevels of prediction. If MUC7 mucin concentration is dropped from theequation, the R² falls to 0.421, even though its influence amounts toonly 17.2% of the outcome.

One object of this study included screening with additional lectins tofind a substitute for MUC7 mucin. Success in employing only lectins andfixed variables for the test allowed for the exploration of the use of asingle reporter solution for reacting the strip that is composed of amixture of the important lectins, calibrated to proportions that areequivalent to their contribution to the regression equation. If alllectins are conjugated to the same reporter molecule, the intensity ofthe dot should be equivalent to their sum. Further calibration achievesthe appropriate responses within the range of the test.

There are many other options available for possible design of the striptest. For example, the inventors have shown that at least for youngadult Caucasians, MUC7 mucin concentration can be used alone and satisfythe goal of three significant non-overlapping zones of prediction. Thisembodiment was designed for the whole saliva/dot blot format by using anantibody to MUC7 mucin sandwiched with the reporter. Different tests canbe specifically designed and calibrated for gender, age, and ethnicityor race. For example, MAL I can be used to predict caries risk in bothCaucasians and Hispanics. Preferably, the test is universalized as muchas possible to avoid the problem that many individuals are a blend ofethnicities or races.

Another strategy that can be used to achieve the goal of threenon-overlapping significant zones of prediction with the wholesaliva/strip test format is the design of the regression analysis.Because caries-free has no variation, the test includes a reporterthreshold above or below which reside all of the caries-freeindividuals. This group is automatically placed into the low caries riskgroup. The remaining individuals are then statistically grouped intomedium or high-risk profiles. The value of this approach is that it is arealistic approximation of this age group, and the statistical demandsfor achieving the remaining two non-overlapping zones of significanceare greatly relaxed and much easier to achieve.

a. Capture strategies. One approach to development of a strip test forcaries uses capture strategies. This involves covalent attachment of anantibody or lectin to the strip, incubating with the saliva sample,rinsing and then reacting the strip with a second antibody or lectinlinked to a reporter. The advantage of this approach is that the testcan be developed with the option of two levels of specificity. Forexample, in one embodiment an antibody directed to the non-glycosylated3′ end of MUC7 can be used for capture, which then allows for probingthe mucin with any of the lectin-reporter complexes that might havepredictive potential. In another embodiment, the apomucin portion of thecaptured mucin is quantitated with a second antibody which is directedat the non-glycosylated 5′ end of the mucin. In yet another embodiment,all saliva components that have a particular oligosaccharideconfiguration are captured using a lectin and then probed with a secondlectin coupled with the reporter. For example, MAL I and MUC7 mucinalong with the three fixed variables (age gender and ethnicity) gave anR² of 0.891 for members of the saliva panel.

b. Test disclosants. The goal for this part of the process was todevelop a one-step procedure that involves a single incubation of thedried spot of saliva with a lectin-reporter complex, two rinses withwater or buffer, and visual comparison of the saliva spot with a pair ofcalibrated standards. The fluorescent spots shown in FIG. 6 demonstratethe success of this protocol. The biotin and avidin complexes werecombined and the resulting biotin-avidin complex was purified beforeincubation with the saliva blot. The fluorescent reporter was AMCA,which is activated by long wave UV and fluoresces in visible blue light.This is a useful reporter because long wave UV lamps are common andinexpensive (black light).

Alternatively, the strip test is evaluated by an enzymatic reaction,such as alkaline phosphatase or horseradish peroxidase. In one example,avidin conjugated to alkaline phosphatase was used for the assessment ofthe saliva panel with lectins, as shown in FIG. 1 and Tables 2 and 3.

The tests strips of this invention have broad application in the fieldof strip tests and other visually oriented diagnostic tests. In oneembodiment, reporters can be used that alter the local pH and thenassess the relative quantity of the reporter with a pH indicator. Forexample, protein derivatizing agents have been identified that addcarboxyl groups to proteins. These derivatizing agents can be used toadd carboxyl groups to the avidin that reacts with the lectin-biotincomplex. When a drop of pH indicator is added, the color change can benoted, which is relatively proportional to the amount of bound avidin.

c. Test devices. In one embodiment the high throughput (HTP) test systemis PAGE-based, which has both analytical and visual capabilities. Inanother embodiment, the high throughput analytical application issimilar to the strip test except that many different saliva samples arepattern spotted on a nitrocellulose surface, communally reacted with thecombination of the lectin-reporters, communally washed and reacted witha color- or fluorescence-generating signal. The actual quantitation isaccomplished with a plate reader such as used for interpreting arrays.For example, the lectin-AMCA complex is suitable for this applicationbecause the quantitation can be judged against the blue colorintensities of a pair of standards, high and-low thresholds, revealed bylong wave UV.

d. Test kits. Test kits provide the ability to validate the test in thegroup and to resolve potential issues that may be dependent onethnicity, races, or ages, etc. Suitable formats for the test kitsinclude the dot blot format and the PAGE system. The kit can includesampling devices, a pre-programmed calculator, standard instructions fornon-X-ray dependent diagnosis of DFT, a test detection device, and thesupplies to perform the required number of tests.

EXAMPLE 3 A Lectin-Based Four Level Caries Risk Test

This study demonstrated that certain combinations of lectins, such asMAL I, JAC and SNA, together with gender information, yields a goodcorrelation with DFT in a statistical anaylsis by regression. This modelprovides four statistically different groups: high, medium, low and zeroDFT into which a tested individual can be placed.

1. Saliva Panel-Dot Blot Assay:

a) This study is a continuation with the same subjects listed in table3, using an alternative version of the high through-put assay system(HTP) of this invention. A notable advance from this study was theachievement of an all lectin system, i.e., MAL I, JAC, and SNA. Whencombined with age and ethnicity, this system forecasts DFT (decayed andfilled permanent teeth) in the ages 7-26 years-old with an R² of 0.926(FIG. 7). While this accomplishment is interesting, it does not have thepotential for risk assessment because it tends to put DFT in numericalorder rather than to age-appropriate categories. For instance, Agingsubject #8 (Table 3) at age 26 with five DFT is in the correct numericalorder above Griffin subjects #3 and #4 (Table 3), ages 7 and 8, withfour DFT each, though Aging subject #8 (Table 3) is in the low to mediumDFT range as an adult and the children appear to be in the highest groupfor their age. In this and the following analysis, MAL I accounts formore than 50% of the regression equation.

b) In modeling for a system that might be compatible with the earlieryoung adult study, with high, medium, and low significant ranges, theinventors found that by pairing the same panel of three lectins (MAL I,JAC, and SNA) and gender, an R² of 0.957 could be achieved (FIG. 8).Interestingly, the model suggests that there are four statisticallydifferent groups: high, medium, low, and very low or zero DFT. Thepossibility of a significant zero category was suggested earlier in themucin data where the low range of the three-level risk model encompassedthree to eight DFT, leaving the zero to two DFT category vacant. Therewere no subjects with zero DFT in that subject group. In the presentlectin-based analysis providing this example, USC subject #42 is 23years-old with zero caries. This subject's derived numerical value,which uses an age-independent regression equation, is significantlylower than the three children, Griffin subjects #10, #13, and #15 (Table3), who also have no caries. The regression analysis numerically placesthese children in a low risk range rather than the zero group,indicating that they will acquire between 2 and 4 DFT by approximately25 years of age. Another interesting forecast is that Griffin subject#5, a seven year-old with one DFT, actually belongs in the medium riskgroup (not low), and based on the inventors′ earlier study, may beexpected to acquire a total of 5 to -8 DFT by 25 years of age. Thechildren in the high risk group are predicted to accumulate 9 or morecaries by the age of 25. One conclusion from this study is that with anappropriate age-independent regression equation, predictions in childrencan be made that forecast the number of caries they will acquire by thetime they are young adults, if individualized preventive treatments arenot applied.

c) Western blots with the lectins MAL I, JAC, and SNA are shown in FIG.9, 10 and 11. The salivas selected for the Western blots represent theextreme combinations of mucin, MAL I, and DFT from the saliva panel. Theblot reacted with MAL I illustrates several very important findings.First, not only both mucins, but also other saliva proteins appear tocarry the oligosaccharide determinants, even in those salivas with highlevels of mucin and no DFT (Griffin subject #15 and USC subject #42;Table 3). Griffin subject #13 had virtually no MUC7 mucin, no cavities,but relatively high MAL I reactivity. The Western blot indicates thatthe MAL I-reactive oligosaccharides in this saliva are primarily locatedon salivary glycoproteins intermediate in size between the two mucins,probably the agglutinins. Saliva from Griffin subject #3, with high DFT,also had relatively high mucin levels (Table 3), but it was nearlydevoid of the MAL I-reactive oligosaccharides. Griffin subject #10, withno DFT, had relatively low MAL I reactivity, but appeared to make up forthis lack by an unusually high level of JAC-reactive oligosaccharides.Both the JAC and SNA Western blots further illustrate the broaddistribution of oligosaccharide determinants among salivary proteins, aswell as striking individual differences.

d) Continuing to use this panel of salivas for discovery, assays werecompleted with the following lectins: MAL I, MAL II, SNA, JAC, AAL, AAA,LTL, LPA, MAA, PNA, UEA I, WGA, and PSA. One example of an improvementarising from this set of experiments is that relative to FIG. 7discussed above, when AAL, UEA I and gender are added and SNA is droppedfrom the analysis, the R² improves from 0.926 to 0.990. This study alsoreaffirms our earlier observation that there may be more than one avenueto achieve the goal of obtaining an R² of >0.90. In this example, withregard to FIG. 8 discussed above with its R² of 0.957, a differentcombination of lectins (MAL I, AAL, JAC, LTL, and PSA) together withgender gives a slightly better R² of 0.966.

e) As potential standards for the strip test, fetuin and glycophorinwere tested against all of the lectins. Glycophorin gave a colorreaction with all of the lectins and thus became the standard forcomparison of lectin affinities between different individuals, whereasfetuin gave only sporadic results.

2. Revisit of the Griffin Study With Salivas From 7-10 Year-Olds Usingthe HTP Assay System for MAL 1. JAC, SNA, and UEA 1.

FIG. 12 shows the HTP assay blot for SNA with all of the salivas in thedata set, including a standard curve. The negative image of the grayscale is shown (FIG. 13), from which the average intensity of each spotis obtained using Sigma Scan Pro (SPSS, Inc.). These intensities werethen converted to standardized units based on the standard curvegenerated from a subset of standards on the same blot. Early in the dataanalysis, several factors became apparent that greatly impacted thequality of subsequent data analyses. First, children with uneruptedpermanent molars contributed a lack of direction to the analyses andwere subsequently excluded. Secondly, because of the variation in thenumber of deciduous teeth per individual, a normalized dfs/t wascalculated and found to be a major contributor to all subsequentanalyses. Some of the more informative analyses included thecontributions of dfs/t, age and the various lectins to correlation withDFT or DFS. These analyses suggested that there might be a usefuldistinction between 7 year-olds and 8-10 year-olds. The contribution ofdfs/t to correlation with DFT or DFS declines from 43% in 7 year-olds to31% in 8-10 year-olds.

3. Additional Studies Eith Salivas From 7-10 Year-Olds Using the HTPAssay System for MAL I, JAC, SN, and UEA I.

With regard to the lectins, either MAL I or UEA L, when grouped withgender and dfs/t, yielded promising correlations with DFT, achieving R²of 0.46 and 0.33, respectively. Separation of the two age groups in theUEA I analysis improved both correlations to an R² of 0.86 for 8-10year-olds and 0.43 for 7 year-olds. Separate analyses for the twoethnicities, Hispanic and Chinese (Mandarin-speaking), furtherillustrate the improvement gained by considering the age groups asdifferent. The Hispanics of all ages gave an R² of 0.49 for DFT vs. UEAI, gender, and dfs/t, whereas the 7 year-old Hispanics alone had an R²of 0.88 and the 8-10 year-olds 0.94. The R² for all of the Chinesechildren was 0.45, whereas for 7 year-olds alone it was 0.80. There werenot enough 8-10 year-old Chinese subjects to complete this comparison.Clearly different regression equations apply to the two age groups.While not wishing to be bound by any particular theory, the inventorsbelieve this reflects the relatively rapid decline in contribution bydfs/t with age and the difference in length of exposure of the permanentteeth.

4. Conclusions

a) The caries test can be performed entirely with lectins.

b) The test can be “universalized” by using lectins as evidenced by thework to date with the saliva panel that represents a microcosm of avariety of races and ages.

c) The use of an age-independent regression equation was alsodemonstrated on the saliva panel. In spite of current DFS, DFS/T, or DFTthat were to low to support assignment of risk levels, the childrenincluded in the saliva panel could be assigned to risk groups based onnumerical values derived from this regression equation, which wasinfluenced heavily by the presence of adults in the analysis. Inaddition, these risk group assignments provide concrete targets of DFSor DFT that will be reached in adulthood if preventive treatments arenot initiated.

d) The companion study, employing young adults, confirmed all of theelements of the 1991 Caucasian study with regard to the correlation ofsaliva mucins and caries history.

e) The companion study also confirmed that different races may havedifferent relationships of mucins to DFS, as first observed in theGriffin Study of 7-10 year-olds, but that use of lectins in the cariestest abolished these differences.

f) Based on the results with the saliva samples from children, it can beconcluded that lectin studies with young adults are the source of theage-independent regression equation(s) that can then be applied tochildren to predict their risk levels.

EXAMPLE 4 Caries Assessment and Risk Evaluation of Permanent Teeth inYoung Adults

The dot blots in this study were performed as described in Example 3.This study included more than 70 young adults ranging in ages from 18 to34 years. The results indicate that of this group, those 24 and olderprovide a subject pool in which age was not a factor in the analyses ofthe relationship of lectin affinities in saliva and caries history.Thus, the 24-34 age group was chosen to represent an end-point of thechildhood to young adult caries acquisition process in permanent teeth.This age group then also provides the end-point for predictions made onyounger ages and the ranges of DFS at each risk level. For FIGS. 14 and15, resting saliva samples were collected from 21 subjects of differentraces and genders. Their ages ranged from 24 to 34 years old. TheirDFS's ranged from 0 to 36. More than 30 different lectin affinities weresampled.

A revealing observation was that intermediate in the study, threelectins (AAL, LTL, and UEA I) combined to create a highly significant(p=0.006) correlation with DFS with an R² of 0.51. The significance ofthis observation is that each lectin is related, to various degrees, tothe secretor status of the individual. However, as more oligosaccharidemotifs were included in the analysis, the role of secretor status becameless prominent and ultimately appeared to have a relatively small (about10%), but significant (p=<0.001), contribution to a complex web ofpositive/negative correlations. AAL, LTL, and UEA I also figuredprominently in all of our previous studies.

All of the sugar affinities, including secretor status, that arerepresented in the above mixture of lectins have previously beenimplicated in the binding or aggregation of various oral microbes(Sharon, N., Adv. Exp. Med. Biol. 408:1-8 (1996)).

FIG. 14 shows the relationship of the caries test results to eachindividual's accumulated caries history. In FIG. 14 the solid line is aplot of the regression equation using DFS as the dependent variable. Thequantitated affinities of 19 different lectins were used in theregression equation. The R² (Coefficient of Determination) is 1.00. Thesignificance is p=<0.001. The dashed lines enclose the PopulationConfidence Interval at 98%, which is also known as the ConfidenceInterval of Prediction.

FIG. 15 shows the assignment of risk levels to different ranges of DFS,and the ability of the lectin-based test to yield a robust regressionequation using risk levels as the dependent variable. For FIG. 15, thesignificance (p) and Confidence Interval of Prediction are the same asfor FIG. 14, however R² is slightly less at 0.998. A modifiedcombination of lectin affinities was also needed in order to achieve the“best” results for FIG. 15. The ranges of DFS that were selected were0-2 DFS for very low risk, 3-8 DFS for low risk, 9-16 DFS for mediumrisk, and 17 or more DFS for high risk. Statistically valid results wereobtained with as many as 12 different risk levels.

EXAMPLE 5 Caries Assessment and Risk Evaluation of Deciduous Teeth inChildren

The focus on the 7-10 year-olds in this study is very important forthree reasons. First, this is the usual time that sealants are appliedto the permanent molars, and the results of the caries risk test canprovide the rationale for application of this preventive treatment on anindividual basis. Secondly, this is the age where statistical correlatesbetween caries history (DFS/T) or risk level in permanent teeth and thecaries assessment test first become evident and can be linked withspecific and predictable. outcomes in young adults. Finally, this agegroup represents an end-point of caries history for the deciduous teeth.

The dot blots in this study were performed as described in Example 3.The number of children in this group is 27, with an approximate equaldistribution of males and females and Hispanic and Chinese, ranging inages from 7-10 years. Though the number of remaining deciduous teethvaries among individuals, the accumulated caries history of thedeciduous teeth is represented in this age group as the end-point ofcaries development in deciduous teeth. FIGS. 16 and 17 illustrate thatthe caries test can accurately assess the number of remaining caries,and translates that into statistically significant risk levels.

FIG. 16 shows the relationship of the caries test results to eachindividual's accumulated caries history. FIG. 16 uses dfs/t as itsdependent variable in order to accommodate the individual differences inthe number of remaining deciduous teeth. The range of remaining carieswas from 0 per tooth to 3 per tooth. The test itself required the inputof 21 different lectin affinities. The R² is 0.996 with a significanceof p=0.001. The Confidence Interval of Prediction was maintained at 98%.FIG. 17 shows the assignment of risk levels to different ranges ofdfs/t, and the ability of the lectin-based test to yield a robustregression equation using risk levels as the dependent variable.

FIG. 17 illustrates the ability of the′ caries test to translate thecaries assessment information into four risk levels based ranges ofdfs/t. Though the Confidence Interval of Prediction at 98% is broaderthan in FIG. 16, there are still four non-overlapping zones ofsignificance that assign all of the subjects into their appropriate risklevels. The regression equation graphed in FIG. 17 has an R² of 0.980,and is significant at p=<0.001. The ranges of dfs/t that were selectedwere 0-0.35 dfs/t for very low risk, 0.36-1.35 dfs/t for low risk,1.36-2.00 dfs/t for medium risk, and 2.01 dfs/t or more for high risk.

EXAMPLE 6 Caries Assessment and Risk Evaluation of Permanent Teeth in aMixed Population of Children and Young Adults

This study provides a model for prediction of future caries developmentin children. The dot blots in this study were performed as described inExample 3. The test group was comprised of 12 subjects, half of whichare young adult Asians and Caucasians and the other half is dividedbetween Chinese and Hispanic 7-9 year-old children. There wereapproximately equal numbers of males and females. The age range of thegroup was 7-26 years-old. FIG. 18 was derived from a regression equationthat included age of the individual, as well as the results of thelectin-based tests as independent variables. Comparison of FIG. 18 withFIG. 7 shows the improvements embodied in the caries risk test over timein a mixed population of children and adults.

FIG. 18 shows that when age is factored in as an independent variable,the lectin-based caries test can accurately assess DFS in a broad rangeof ages. The rationale for prediction of future caries development isbased on the idea that risk levels are first derived from a young adultgroup of subjects that represents an end-point for caries development,such as was done in FIG. 14.

FIG. 19 shows that age is no longer a factor and the regression equationwas generated by the lectin-based tests on young adults, as were therisk levels. The lectin-based data for the children was analyzed by theadult-derived equation to yield the observed risk levels. FIG. 19 showsthat when the caries test data from the children is processed by theadult-derived regression equation, the children are then assigned tospecific risk levels. The ranges for the different risk levels in FIGS.18 and 19 are the same as those above, i.e., 0-2 DFS for very low risk,3-8 DFS for low risk, 9-16 DFS for medium risk, and 17 or more DFS forhigh risk. The interpretation from this application of the caries testis that even though some of the children in this group have no caries ontheir permanent teeth, the test suggests that they will acquire 3-8 bythe time they are approximately 25 years-old. The test also suggeststhat the child with one DFS will acquire from 8 to 15 more caries by age25. The high risk children are targeted to accumulate 17 or more DFS bythe age of 25. Comparison of FIG. 19 with FIG. 8 illustrates theimprovement in the prediction potential of the caries risk test.

EXAMPLE 7 Colored Microbead Assays

FIG. 20 illustrates the use of the blue-yellow bead model to expressintermediate ratios of two lectins as green while still preserving theend points of the color scale. LTL and LEL were derivatized directly toblue and yellow microlatex beads, respectively. To accomplish the testitself, the concentrations of derivatized beads were first calibratedseparately on saliva samples that represented the population range ofaffinities observed on the dot blot assay. This is shown on the top twotests of FIG. 20. The beads were then mixed to achieve the calibratedconcentrations, and the suspension was overlaid on the membranecontaining 5 mL spots of dried saliva. Binding was rapid-and appeared tobe limited by bead settling times.

The bottom of FIG. 20 shows the full range of colors. In the regressionequation that is illustrated with these saliva samples, LTL ispositively correlated with DFS, and LEL is negatively correlated withDFS. In FIG. 20, the numbers represent saliva samples from differentstudents. The colors coincide with the following DFS for this group:#2-34 DFS, #69-20 DFS, #70-17 DFS, and #107-5 DFS. Student #2 has a veryhigh ratio of LTL to LEL affinities, but even at low concentrations ofeach, the color on the test strip is blue. Students #69 and #70 alsohave relatively high rations, >10:1, but since the test was beencalibrated to see this as an intermediate ratio, the color on the teststrip is green. Student #107 has a ratio of 0.46:1 of LTL:LEL, i.e., avery low ratio of LTL to LEL, and the color on the test strip is yellow.The color test appears to preserve the ratio of positive to negativeaffinities, in spite of over-all concentration differences betweensaliva samples.

As an alternative, the red-yellow combination was also tested with thesame lectins and saliva samples. Like the above test, the two end colorsformed the intermediate color (orange), which dominated at most ratiosof the two lectins (FIG. 21). A weakness of both color combinations isthe difficulty of visualizing the pure yellow color against the whitebackground. As an alternative, LEL was conjugated to a mixture of redand yellow beads, producing the easier-to-see orange. The most recentcolor endpoints are blue and orange with intermediate colors that arestill predominantly shades of green. As noted above, each test will havea range of standards covalently linked to the test strip to produce thedifferent colors associated with each risk level.

EXAMPLE 8 Permissive and Preventive Oligosaccharide Motifs Suggested bythe Lectin Affinity Studies

This study was to determine the most important oligosaccharide motifssampled for the test. The strategy was to search for the convergence ofcorrelations between lectin affinities in the context of theirindividual significant positive or negative correlations with DFS usingthe most recent group of student saliva samples.

The two main core motifs that emerged are Fucα-1,2Gal-GlcNAc (also knownas the H antigen, which is usually associated with the secretor positivetrait) and ±Galαorβ-1,3GalNAcαorβ. In short, these oligosaccharidemotifs when not sialylated were permissive, i.e., positively correlatedwith DFS, but when sialylated either by α-2,3 or α-2,6 sialic acid, theywere preventive,.i.e., negatively correlated with DFS. The one exceptionis that ±Galα-1,3GalNAcβ also appears to be preventive whether or not itis sialylated. The third motif that appears to be important in the testis chitobiose or chitotriose (GlcNAc_((2 or 3))). This also shifted frompermissive to preventive upon sialylation. In addition, when thechitobiose segment is α-fucosylated, which is likely to include theLewis^(a) antigen, it appears to be preventive regardless ofsialylation.

It is also noteworthy that while six of the 21 students in the 24-34year-old group are non-secretors as judged by very low levels of UEAaffinities, the H antigen motif was equally important to the testregardless of its absolute concentration even in the non-secretors, allof whom still had measurable amounts of UEA affinity binding. Thisanalysis affirms that the ratios of lectin affinities are more importantto the test than the absolute affinity-based concentrations.

The foregoing description is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and processshown as described above. Accordingly, all suitable modifications andequivalents may be resorted to falling within the scope of the inventionas defined by the claims that follow. The words “comprise,”“comprising,” “include,” “including,” and “includes” when used in thisspecification and in the following claims are intended to specify thepresence of stated features, integers, components, or steps, but they donot preclude the presence or addition of one or more other features,integers, components, steps, or groups thereof.

1. A method for predicting the risk of dental caries in a subject, saidmethod comprising: providing an unfractionated saliva sample from saidsubject; contacting an aliquot of said saliva with one or more lectinsunder conditions that allow said one or more lectins to bind to one ormore lectin-binding components of said saliva; detecting the amount ofbound lectin; and comparing the amount of bound lectin to the amountknown to bind a saliva sample from a control subject, wherein the amountof bound lectin is indicative of the risk of dental caries.
 2. Themethod of claim 1, wherein said saliva sample is an unstimulated salivasample.
 3. The method of claim 1, wherein said lectin-binding componentis an oligosaccharide.
 4. The method of claim 1, wherein said lectin isselected from the group consisting of DSL, ECL, PSA, WGA, UEA, MALI,MAA, PNA, AAL, LTL, MAL II, JAC, LEL, SNA, PTL I, ACL, GSL II, VVA,BPL,WFL, SJA, MPL, GNL, HHL, CCA, NPL, STL, PHA-L, PHA-E, GSL I, DBA, HMA,EEA, LPA, and PTL II.
 5. The method of claim 1, wherein said lectin isnot PNA.
 6. The method of claim 4, wherein said lectin is MALI.
 7. Themethod of claim 1, wherein said one or more lectins are selected fromthe group consisting of AAL, LTL and UEA
 1. 8. The method of claim 1,wherein said one or more lectins are selected from the group consistingof DSL, ECL, PSA, MAL I, PNA, AAL, LTL, MAL II, JAC, LEL, PTL I, GSL II,VVA, BPL, SJA, MPL, and CCA, and said subject is an adult.
 9. The methodof claim 1, wherein said one or more lectins are selected from the groupconsisting of ACL, PNA, LTL, PSA, MAL II, MAA, STL, PTL I, LEL, DSL,ECL, AAL, VVA, GNLI, CCA, SNA, JAC, WFL, SJA, MALI, and BPL, and saidsubject is a child. 10.-14. (canceled)
 15. The method of claim 1,wherein said contacting comprises: applying a drop of said saliva to amatrix material; and contacting the matrix with a solution containingsaid one or more lectins.
 16. The method of claim 1, wherein said one ormore lectins are coupled to a reporter selected from the groupconsisting of dyes, chemiluminescent compounds, enzymes, fluorescentcompounds, biotin, haptens, radioluminescent compounds, andradioactive-labeled biomolecules. 17-25. (canceled)
 26. The method ofclaim 1, wherein said subject is a human. 27-28. (canceled)
 29. Themethod of claim 1, wherein said dental caries is selected from the groupconsisting of early-onset dental caries, adult dental caries, rootcaries, DFT, DMF, DMFS, dfs, dft, dmft, dmfs, and dfs/t. 30-47.(canceled)
 48. A method for preventing or reducing the risk of dentalcaries, comprising: providing an unfractionated saliva sample from asubject; contacting an aliquot of said saliva with one or more lectinsunder conditions that allow said one or more lectins to bind to alectin-binding component of said saliva; detecting the amount of boundlectin; comparing the amount of bound lectin to the amount known to binda saliva sample from a control subject,. wherein the amount isproportional to the risk of dental caries in said subject; andadministering a therapeutic reagent to said subject when the content ofsaid component in said saliva is above or below the level contained in anormal control.
 49. A kit for detecting dental caries comprising: meansfor collecting a saliva sample; means for measuring the amount of alectin-binding component in said sample; and an oral fluid standard forcomparing the amount of said component in said sample. 50-53. (canceled)54. The kit of claim 49, wherein said dental caries is selected from thegroup consisting of early-onset dental caries, adult dental caries, rootcaries, DFT, DMF, DMFS, dfs, dft, dmft, dmfs, and dfs/t.
 55. The kit ofclaim 49, which is a Western blot format.
 56. An assay device fordetecting the presence of lectin-binding components in a saliva sample,said device comprising: a sample receiving zone comprising a firstmatrix material for receiving an aliquot of said sample; and a controlzone comprising a second matrix material having at least one controllectin-binding compound of a known concentration bound to the surface ofsaid second matrix material.
 57. The device of claim 56, wherein saidsample receiving zone matrix material is selected from the groupconsisting of nitrocellulose, cotton, polyester, rayon, nylon,polyethersulfone, and polyethylene.
 58. The device of claim 56, whereinsaid sample receiving and control zones are affixed to the top side of asemi-rigid support.
 59. The device of claim 58, wherein semi-rigidsupport comprises polypropylene, poly(vinyl chloride), propylene, orpolystyrene.
 60. An assay device for detecting the presence oflectin-binding components in a saliva sample, said device comprising: asample receiving zone comprising a first matrix material and one or morelectins bound to said matrix material; and a control zone comprising asecond matrix material and having at least one control saliva sample ofa known concentration.